Design, synthesis and evaluation of aminobenzophenone derivatives containing nitrogen mustard moiety as potential central nervous system antitumor agent

Article abstract of DOI:10.1007/s00044-013-0582-8

A series of novel substituted aminobenzophenone derivatives containing nitrogen mustard moiety (5a-f) were synthesized and characterized on the basis of their IR, ¹H NMR, ¹³C NMR, CHN, and mass spectral data. All the compounds when evaluated for chemical 4-(4-nitrobenzyl) pyridine alkylating activity proved to be active alkylating agents. All the synthesized compounds were subjected to physicochemical parameters determination required for central nervous system (CNS) activity through computational, online software, and QikProp 3.2. The log P values and other in silico ADME physicochemical descriptors analyzed lay between the ranges those are required for good BBB penetration. The in vitro antiproliferative activity against human cancer cell lines viz. A 549 (lung), COLO 205 (colon), U 87 (glioblastoma), and IMR-32 (neuroblastoma) was investigated. Most of the test compounds showed potent antitumor activity, especially compound (5f) which displayed the highest activity against CNS cancer cell line comparable to that of chlorambucil and docetaxel. The preliminary structure-activity relationship (SAR) revealed that 5-chloroaminobenzophenone-mustard series (5a-c) exhibited better antitumor activity than 5-nitroaminobenzophenone-mustard series (5d-f).

Full text of DOI:10.1007/s00044-013-0582-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0582-8
ORIGINAL RESEARCH
Design, synthesis and evaluation of aminobenzophenone
derivatives containing nitrogen mustard moiety as potential
central nervous system antitumor agent
•
Rajesh K. Singh D. N. Prasad T. R. Bhardwaj
•
Received: 4 December 2012 / Accepted: 13 March 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of novel substituted aminobenzophe-
none derivatives containing nitrogen mustard moiety
(5a–f) were synthesized and characterized on the basis of
their IR, 1H NMR, 13C NMR, CHN, and mass spectral data.
All the compounds when evaluated for chemical 4-(4-
nitrobenzyl) pyridine alkylating activity proved to be active
alkylating agents. All the synthesized compounds were
subjected to physicochemical parameters determination
required for central nervous system (CNS) activity through
computational, online software, and QikProp 3.2. The log
P values and other in silico ADME physicochemical
descriptors analyzed lay between the ranges those are
required for good BBB penetration. The in vitro antipro-
liferative activity against human cancer cell lines viz. A
549 (lung), COLO 205 (colon), U 87 (glioblastoma), and
IMR-32 (neuroblastoma) was investigated. Most of the test
compounds showed potent antitumor activity, especially
compound (5f) which displayed the highest activity against
CNS cancer cell line comparable to that of chlorambucil
and docetaxel. The preliminary structure–activity rela-
tionship (SAR) revealed that 5-chloroaminobenzophenone-
mustard series (5a–c) exhibited better antitumor activity
than 5-nitroaminobenzophenone-mustard series (5d–f).
Keywords 2-Aminobenzophenone ꢀ Nitrogen mustard ꢀ
Blood–brain barrier ꢀ Physicochemical ADME descriptors ꢀ
NBP assay ꢀ In vitro cytotoxicity
Introduction
Cancer is considered as the most serious health problem all
over the world. Of all the cancer, Brain cancers are the sec-
ond leading cause of cancer-related deaths in males while the
fifth leading cause of cancer-related deaths in women. It
accounts for 85–90 % of all primary central nervous system
(CNS) tumors. Approximately, 42 % of all brain and CNS
tumors occurred in males and 58 % in females (Dolecek
et al., 2012).
Nitrogen mustard classes of drug such as chlorambucil
and mechlorethamine, bifunctional alkylators are still used
extensively in the treatment of leukemia and solid tumors.
The main demerits of mustard drug are their high reactivity
so toxic to normal peripheral tissue and hydrophilicity
which does not allow the molecule to cross blood–brain
barrier (BBB) and, therefore, make these drugs not of much
clinical significance to patients suffering from brain tumor
(Francisco et al., 2008). Various attempts have been made
to overcome the limited access of nitrogen mustard into the
brain such as synthesizing their lipophilic analogs (Genka
et al. 1993) or by linking the active anticancer moiety to
lipophilic carrier (Peng et al., 1975; Hirata and Driscoll,
1976; Bartzatt, 2004) and chemical drug delivery system
(Bodor et al., 1989; El-Sherbeny et al., 2003). Moreover,
recent study revealed that targeting N-mustard moiety by
linking to various carriers having optimized physico-
chemical properties resulted in more potency and less
peripheral cytotoxicity than the corresponding untargeted
mustard (Mourelatos et al., 2012; Marvania et al., 2011;
Design, synthesis and evaluation of aminobenzophenone derivatives
containing nitrogen mustard moiety as potential central nervous
system antitumor agent.
R. K. Singh (&) ꢀ D. N. Prasad
Pharmaceutical Chemistry Division, Shivalik College
of Pharmacy, Nangal, Rupnagar 140126, Punjab, India
e-mail: rksingh244@gmail.com
T. R. Bhardwaj
Indo-Soviet Friendship (ISF) College of Pharmacy, Moga
142001, Punjab, India
123

Med Chem Res
Scutaru et al., 2011; Kapuriya et al., 2011; Zheng et al.,
2010; Reux et al., 2008; Fousteris et al., 2007). So, the aim
of the present investigation is to improve N-mustard
physicochemical properties by attaching it chemically to
CNS active agent to obtain drug capable of penetrating the
brain.
the phenyl ring A at position 7 [Cl, NO2] and the phenyl
ring C at position 2 and 4 [Cl] to study the effect of the
substitution at these positions on the antitumor activity
against four cell lines.
2-Aminobenzophenone derivatives are important com-
pounds in organic chemistry because of their application in
heterocyclic synthesis and medicines (Walsh, 1980). The
2-Aminobenzophenones have been used as starting mate-
rial for the synthesis of various 1, 4-benzodiazepines hav-
ing potent CNS therapeutic activity (Sternbach et al.,
1962). It has also been used for the synthesis of pepti-
doaminobenzophenones, a novel class of ring open deriv-
atives of 1, 4-benzodiazepines, evaluated for CNS activity
(Hirai et al., 1980; Hirai et al., 1981). Recently, 2-amino-
benzophenone derivatives were reported to exhibit anti-
mitotic (Liou et al., 2002), p 38 MAP Kinase Inhibiting
activity (Ottosen et al., 2003) and a novel class of brady-
kinin B1 receptor antagonists activity with good brain
penetration (Su et al., 2008). 2-Aminobenzophenone
derivatives were considered of our interest because they
fulfill all the structure requirements as the benzodiazepine
nucleus contains. According to SAR study, electron with-
drawing substituents at ring A and B are important for the
CNS activity of benzodiazepines as shown (Fig. 1).
Prompted by these claims, we assumed that incorpo-
rating these potent pharmacophores of benzodiazepine
nucleus responsible for CNS action together with N-mus-
tard moiety may result in strong anticancer agent that may
pass BBB and effective against brain tumor.
Results and discussion
Chemistry
The target aminobenzophenone-mustard derivatives (5a–f)
were synthesized as shown in Scheme 1. The starting
compounds (3a–f) were prepared according to the previous
reported methods (Sternbach et al., 1963). Treatment of
(3a–f) with chloro/bromoacetylchloride in toluene under
conventional and microwave irradiation, gave products
(4a–f). Microwave method was found to give better yield
in less time. For the representative compound 4a, the
characteristic IR spectra showed the disappearance of band
of –NH2 at 3,400 and 3,340 cm-1 and appearance of single
band at 3,280 for secondary amide (–NHCO). The
appearance of significant band at 1,646 cm-1 assigned to
carbonyl of –NHCO further indicates the formation of 4a.
1
In HNMR spectra, the appearance of (COCH2Cl) protons
as singlet at d 4.0 ppm and (CONH) protons exchangeable
to D2O at d 11.0 ppm as broad singlet confirms the for-
mation of compound 4a Table 1.
Target compounds (5a–f) were synthesized by two
methods to optimize the yield and the purity. In method A,
(4a–f) was treated with diethanolamine and then chlori-
nation by phosphorous oxychloride to get desired com-
pounds in 50–60 % yield. In order to increase the yield by
reducing steps, (4a–f) were directly condensed with bis (2-
chloroethyl)amine hydrochloride. But, the yield was even
less (25–35 %) (Table 2). This is due to high reactivity of
–Cl group and hence multiple side products may have
In continuation of our efforts for the synthesis of CNS
active therapeutic agent (Singh et al., 2011, 2012a, b, c,
2013), in the present work, new substituted 2-aminobenz-
ophenones derivatives (5a–f) were synthesized, incorpo-
rating the N-mustard group and varying the substitution on
CNS activity decreases
with large alkyl group
H
N
O
C
R1
O
N
Cl
Cl
Electron withdrawing group
A
at 7 increases the CNS activity
A
N-Mustard
moiety
R
C
H3C
N
O
R
N
B
X
X
B
PROPOSED STRUCTURE
CNS activity increases
by electron withdrawing
group
Fig. 1 Design of aminobenzophenone-mustard anticancer agents
123

Med Chem Res
NH2
COCl
NH2
O
R1
+
R
(i)
R1
R
R2
(1)
(2)
R2
(3a-f)
(iia)
(iib)
O
O
Cl
X
N
N
N
Method A
H
O
H
O
Cl
X= Cl, Br
Method B
R
R
R1
R1
R2
R2
(5a-f)
(4a-f)
Where
R
Cl
Cl
Cl
NO2
NO2
NO2
R1
H
Cl
H
H
Cl
H
R2
H
H
Cl
H
5a
5b
5c
5d
5e
5f
H
Cl
Scheme 1 Reagents and conditions (i) Zinc Chloride, (iia) Conventional, (iib) Microwave, ClCH2COCl, BrCH2COBr (iii) Method A:
Diethanolamine, DMF, NaI, POCl3, Method B: Bis(2-chloroethylamine)hydrochloride, K2CO3/NaI, DMF
Table 1 Comparison of % yield and time taken by compounds 4(a–f)
from both methods
Table 2 Comparison of % yield by methods A and B for the syn-
thesis of compounds (5a–f)
S.
No.
Compounds m.p.
(°C)
Conventional
Microwave
S. No. Compounds m.p. Method A % Yield Method B %Yield
Yield
(%)
Time
(h)
Yield
(%)
Time
(min)
1.
2.
3.
4.
5.
6.
5a
5b
5c
5d
5e
5f
Oil 52
Oil 54
Oil 52
Oil 58
Oil 60
Oil 57
25
29
28
32
35
32
1
2
3
4
5
6
4a
4b
4c
4d
4e
4f
118–120 80
132–134 67
2.5
3.0
4.0
3.5
2.5
3
94
88
85
85
82
78
1.0
2.5
3
126–128 65
154–156 66
146–148 70
156–160 63
2.0
1.5
2.5
which is more deshielded due to the electronegativity effect
of chlorine.
formed. For the series of aminobenzophenone-mustard
derivatives (5a–f), the 1H NMR spectra of these com-
pounds shows the ethylene bridge yielding two triplets
integrating for four protons each. One triplet appeared at d
3.00–3.75 ppm which was assigned to –CH2 group adja-
cent to nitrogen. The other triplet at d 3.5–4.6 ppm was
assigned to the –CH2– group adjacent to the –Cl group
NBP alkylating activity assessment
The final compounds were evaluated by its alkylating
activity using 4-(4-nitrobenzyl) pyridine (NBP) as an ana-
lytical reagent. It is hypothesized that there is correlation
between the chemical alkylating activity and anti-tumor
123

Med Chem Res
differentiates between the reactivities of the synthesized
compounds (5a–f) under specified conditions using chlor-
ambucil as positive control. All the compounds proved to be
active alkylating agents (Table 3).
In silico physicochemical evaluation
Target compounds were designed to be CNS active; hence,
the parameters were selected which affect BBB. Lipo-
philicity (logP), hydrophilicity (logS), and logBB were the
principle descriptors to be identified as important for CNS
penetration. Table 4 presents the partition coefficient val-
ues for synthesized derivative (5a–f) by computational,
online software, and by QikProp. All the values are within
required range indicating optimum lipophilicity. In addi-
tion, a compound possessing tertiary nitrogen (a feature of
many CNS drugs) shows a higher degree of brain perme-
ation (Pajouhesh and Lenz, 2005). All the compounds
(5a–f) have tertiary nitrogen in their structure and hence
indicate BBB permeability of the synthesized compounds.
Other physicochemical descriptors obtained by QikProp
are presented in Table 5 support the clinical potential of
these synthesized compounds. The physically significant
descriptors of mechlorethamine, chlorambucil, and ami-
nobenzophenone-mustard derivatives (5a–f) were analyzed
for the CNS activity and drug-like properties. Polar Surface
Area (PSA) has become an important molecular descriptor
for drug absorption and blood–brain barrier penetration
(Clark, 1999, Kelder et al., 1999). The values of PSA for
all synthesized compounds (5a–f) are within the range that
indicates good penetration of the blood–brain barrier.
QPPMDCK predicted apparent MDCK cell permeability
in nm/s. MDCK cells are considered to be a good mimic
for the blood-brain barrier (Wang et al.,2005). Higher the
value of MDCK cell, higher the cell permeability. All the
target compounds (5a–f) have QPPMDCK values within
the range and hence good BBB permeation.
Fig. 2 Formation of the aminobenzophenone-mustard (5a–f)–NBP
adduct in acetone/ethanol medium; variation in absorbance (kmax
542–560 nm) with time; compared with chlorambucil
Table 3 The alkylating activity (NBP assay) of the synthesized
compounds (5a–f)
Compounds R
R1 R2 kmax Temp
(°C)
K
t1/2
min-1 9 10-2 (min)
5a
Cl
H
Cl
H
H
H
H
542 65
545 65
5.21 0.156 13.3
6.72 0.156 10.3
7.78 0.058 8.9
5.87 0.175 11.8
7.29 0.127 9.5
8025 0.089 8.4
10.65 0.122 6.5
5b
Cl
5c
Cl
Cl 545 65
5d
NO2
H
H
554 65
558 65
5e
NO2 Cl
NO2
5f
H
Cl 558 65
560 85
Chlorambucil
activity (Gomez-Bombrelli et al., 2012). The reaction was
determined at 65 °C in 50 % aqueous acetone using spec-
trophotometric quantitation. The 4-(4-nitrobenzyl) pyridine
reacts with alkylating agents (5a–f) to form aminobenzo-
phenone-mustard-NBP adduct which gives a purple color
upon basification. The intensity of the produced color mea-
sured by UV spectrophotometer as absorbance is directly
proportional to the degree of alkylation (Fig. 2). This method
Table 4 Various partition coefficient parameters obtained by various tools important for CNS activity
S.No. Compounds miLogPa \ 5 CSLogPb \ 5 ClogPc \ 5 QP logP o/wd (-2.0–6.5) QP logSd (-6.5–0.5) QP logBBd (-3 to 1.2)
1.
2.
3.
3.
4.
5.
a
5a
5b
5c
5d
5e
5f
4.786
5.416
5.464
4.067
4.697
4.745
4.56
4.84
4.9
5.15
5.66
5.91
4.35
4.83
5.09
4.355
4.637
4.876
3.253
3.400
3.580
-3.823
-4.173
-4.690
-3.200
-3.400
-3.900
0.522
0.502
0.658
3.841
4.181
4.481
-0.663
-0.643
-0.494
Calculated by method of Molinspiration
www.molinspiration.com/cgi-bin/properties
b
Calculated by method of Chemsilico
www.chemsilico.com/cs_products/products.html
c
Calculated by ChemDraw Ultra 8.0
d
Calculated by QikProp
123

Med Chem Res
Table 5 In silico Pharmacokinetic parameters with their optimum range important for CNS activity and oral bioavailability obtained by QikProp
tool
Descriptors
MCEa
CBLb
5a
5b
5c
5d
5e
5f
-1
CNS (-2 to ?2)
2
-1
2
2
2
-1
-1
M.WT \ 500
PSA (7.0–200)
156.055
304.216
413.73
448.175
448.175
59.26
0
424.283
458.728
458.728
5.6
55.2
59.28
59.0
103.85
105.8
107.2
Donar HB (0–6)
0
1
0
0
1
1
1
Accept HB (2.0–20)
2
3
5.5
5.5
5.5
6
3.253
8
6
3.40
8
6
3.580
8
QP logP o/w (-2.0–6.5)
Rot bond(0–15)
1.967
4.671
4.355
4.637
4.876
7
2
7
7
7
QP log S (-6.5–0.5)
-1.122
-5.347
227.915
-0.583
802.855
3
-3.823
-4.173
-4.69
754.395
0.658
-3.20
188.64
-0.663
188.6
3
-3.40
216.85
-0.643
216.8
3
-3.90
473.90
-0.494
474.0
3
QPPCaco \ 25 poor [ 500 good
QP logBB (-3.0–1.2)
QPPMDCK \ 25 poor [ 500 good
Metablsm (1–8)
2300.154
754.447
651.918
1.173
0.522
0.502
8513.882
4233.643
5201.516
10000
1
100
1
2
100
4
2
100
4
2
100
4
% Hu Ab [ 80% high \ 25% poor
Nand O \ 2–15[
96.495
3
81.2
7
83.2
7
96.95
7
Violation of rule of five (max 4)
Violation of rule of three (max 3)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
a
Mechlorethamine
b
Chlorambucil
In accordance with Lipkinski’s rule of 5 (Lipinski et al.,
1997), the molecular weights of compounds should have
B500 Daltons, B5 hydrogen bond donors, B10 hydrogen
bond acceptors, log P of B5 and rotatable bond B10.
Compounds that satisfy these rules are considered drug-
like. According to this rule, compounds with number of
violations not more than 1 shows good bioavailability. All
the synthesized compounds (5a–f) have no violation of rule
of five. Compounds with H-bond donor less than 5 and
H-bond acceptors less than 10 shows good BBB penetra-
tion (Osterberg and Norinder, 2000). All the synthesized
compounds have H-bond donor values in the range of 0–1
which are less than 5 and H-bond acceptors value in the
range of 5–6 which are less than 10. Compounds with
molecular weight less than 500 Daltons shows significant
passive lipid-mediated transport through BBB (Leeson and
Davis, 2004). All the test compounds have molecular
weight in the range of 413–458 which is \500. According
to Jorgensen’s rule of three (Jorgensen, 2009), there should
be QPlogS [-5.7, QPPCaco [22 nm/s, # primary
metabolites\7. In accordance with the data obtained from
Table 5, all the compounds have no violation of Jorgen-
sen’s rule of three and hence potential to be CNS active.
In vitro anticancer screening
The newly synthesized compounds were evaluated for their
in vitro cytotoxic activity against human cancer cell lines: A 549
(lung), COLO 205 (colon), U 87 (primary glioblastoma cells),
and IMR-32 (neuroblastoma cell lines) using chlorambucil and
Table 6 The antiproliferative activity data of synthetic compounds (5a–f) assessed by the MTT reduction assay
Compounds
IC50 Values S.D.
R
R1
R2
A549
COLO 205
U87
IMR32
5a
5b
5c
5d
5e
5f
Cl
H
H
37.093 1.63
34.634 1.92
37.336 1.94
64.806 2.33
64.806 2.33
36.673 2.89
27.256 1.75
21.09 1.72
52.283 2.09
32.573 2.70
28.683 2.02
32.896 2.87
33.38 2.19
38.93 1.46
30.706 1.72
26.67 1.09
29.013 1.15
24.53 1.89
27.134 2.33
29.464 1.71
43.744 2.47
23.267 2.29
20.72 1.19
18.82 1.09
29.393 2.03
30.816 3.15
31.134 2.96
33.2 2.27
Cl
Cl
H
H
Cl
Cl
H
NO2
NO2
NO2
H
Cl
H
H
31.396 1.50
28.394 1.67
23.71 1.73
20.09 1.03
Cl
STD DOCETAXEL
STD CHLORAMBUCIL
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 student’s t test. A value of p \ 0.05 was considered as significant
123

Med Chem Res
docetaxel as the reference drugs.The parameter used here is
IC50, which corresponds to the concentration required for 50 %
inhibition of cell viability. The IC50 of the synthesized com-
pounds compared to the reference drug are shown in Table 6.
From the results in Table 6, it was found that most of the test
compounds showed potent antitumor activity comparable to
that of chlorambucil and docetaxel. In general, 5-chlor-
oaminobenzophenone-mustard series (5a–f) exhibited better
antitumor activity than 5-nitroaminobenzophenone-mustard
series (5d–f). Careful examination of the effect of –Cl group on
ring B at o or p position showed that the presence of chlorine at
these positions does not make much difference in activity of the
compounds. Compound 5f was the most active compound in
this study against both CNS cancer cell lines, while, compound
5b and 5c were the most active compounds against A549 and
COLO 205 cell lines, respectively. It is also worthwhile mention
that compounds 5a and 5e were not much active against A549
and COLO 205 cell lines, respectively.
RX-I spectrometer 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 Q-TOF Mass spectrometer. Elemental
analysesforC, H,andNwereperformedonThermo-flashEA-
1112 CHNS-O analyzer. The synthesis related to microwave
irradiation were carried out in domestic LG little chef
microwave oven. The Chromatography was carried out using
Merck silica gel 60 (230–400 mesh). Anhydrous sodium
sulfatewasutilizedasdryingagents. Acomputationalstudyof
titled compounds was performed for prediction of ADME
¨
properties by QikProp 3.2 tool available in Schrodinger 9.0
version and Molinspiration online property calculation tool-
kit. Bis(2-chloroethylamine)hydrochloride was prepared by
thionyl chloride-assisted chlorination of diethanolamine by
reported procedure (Friedman and Seligman, 1954). The
syntheses of substituted 2-aminobenzophenones (3a–f) were
carried out by the literature method (Sternbach et al., 1962).
General method for the syntheses of substituted
2-(chloroacetamido)benzophenones (4a–f)
Conclusion
In conclusion, a series of novel substituted 2-aminobenzo-
phenone derivatives incorporating nitrogen mustard as
cytotoxic moiety were synthesized and were subjected to
physicochemical parameters determination for BBB pene-
tration through online software. The in vitro NBP alkylation
studies revealed that all compounds have active alkylating
activity. Furthermore, a computational study was carried out
for prediction of in silico physicochemical properties
required for CNS activity and drug-likeliness. All pharma-
cokinetic descriptors were within the range set by Lipinski’s
rule of five and Jorgensen’s rule of three and possessing
favorable physicochemical properties for acting as CNS
drugs, making them potentially promising agents for CNS
tumor. The invitro antiproliferative activity of the newly
synthesized compounds against human A 549, COLO 205, U
87, and IMR-32 was investigated. Most of the test com-
pounds showed potent antitumor activity, especially com-
pound (5f) which displayed the highest activity against CNS
cancer cell line comparable to that of chlorambucil and
docetaxel. Further studies are still needed to determine the
invivo studies of the compounds as well as to explore the
SAR of the other positions of the benzene ring.
Conventional method
Compounds (4a–f) were prepared as per the published pro-
cedure (Sternbach et al., 1961, 1963) that involves treating
substituted 2-aminobenzophenones (3a–f) (25 mmol) with
chloroacetyl chloride/bromoacetyl bromide (50 mmol) in
toluene (50 ml) and refluxing for 2 h to expel most of the
formed hydrogen chloride. The solution was then cooled,
washed with ice cold dilute aqueous ammonia solution,
dried, filtered and concentrated in vacuo. The residue was
recrystallized from alcohol to get pure substituted 2-(chloro/
bromoacetamido)benzophenone (4a–f).
Microwave irradiation method
A
solution of substituted 2-aminobenzophenones
(2.5 mmol) and chloroacetyl chloride/bromoacetyl bro-
mide (5.0 mmol) in toluene (5 ml) was irradiated for
1–3 min in microwave oven (360 W). The solution was
then cooled, washed with ice cold dilute aqueous ammonia
solution, dried with anhydrous sodium sulfate, filtered and
concentrated in vacuo. The recrystallization of the crude
residue from alcohol afforded substituted 2-(chloro/
bromoacetamido)benzophenones (4a–f).
Experimental
2-(chloroacetamido)-5-chlorobenzophenone (4a)
The melting pointsweredeterminedonVeego-programmable
melting point apparatus (microprocessor-based) and are
uncorrected. 1H-NMR and 13C-NMR spectra were obtained
usingBrukerAvance-II, 400MHZspectrometer.Infrared(IR)
spectra were recorded on Perkin Elmer model 1600 FT-IR
Anal
IR KBr (cm-1) 3,280 (N–H), 3,059, 2,961 (C–H), 1,692
(C=O), 1,646 (NHC=O), 1,514 (C=C), 1,286 (C–N) and
123

Med Chem Res
752 (C–Cl); 1H-NMR (CDCl3) d (ppm): 4.19 (s, 2H,
-COCH2N), 7.50–7.70 (m, 8H, ArH), d 11.4 (brs, 1H,
exchangeable, -NHCOCH2-); 13C-NMR (CDCl3) d (ppm):
43.3 (C–Cl), 122.4, 125.2, 128.4, 129.6, 130.0, 130.6,
132.8, 132.0, 139.2, 141.0 (Aromatic) 168.0 (NHC=O),
192.8 (C=O); Anal. calcd for C15H11NO2Cl2: C % 58.46,
H % 3.60, N % 4.55; Found: C % 58.36, H % 3.72, N %
4.42.
(C–N) and 628 (C–Br); 1H-NMR (CDCl3) d (ppm): 4.0 (s,
2H, –COCH2N), d 7.17–7.55(m, 7H, ArH), 9.67 (brs, 1H,
exchangeable, –NHCOCH2–); 13C-NMR (CDCl3)
d
(ppm): 31.4 (C–Br), 122.5, 125.3, 125.8, 126.8, 127.2,
128.2, 131.4, 133.8, 136.8, 138.6, 144.3, 148.6 (Aromatic),
164.8 (NHC=O), 197.8 (C=O); Anal. calcd for
C15H10N2O4BrCl: C % 45.31, H % 2.54, N % 7.05;
Found: C % 45.38, H % 2.62, N % 7.12.
2-(chloroacetamido)-5, 20-dichlorobenzophenone (4b)
2-(bromoacetamido)-40-chloro-5-nitrobenzophenone
(4f)
IR KBr (cm-1) 3,284 (N–H), 3,065, 2,958 (C–H), 1,689
(C=O), 1,652 (NHC=O), 1,518 (C=C), 1,290 (C–N) and
757 (C–Cl); 1H-NMR (CDCl3) d (ppm): 3.8 (s, 2H,
-COCH2N), 7.40–7.60(m, 7H, ArH), 11.3 (brs, 1H,
exchangeable, -NHCOCH2-); 13C-NMR (CDCl3) d (ppm):
43.0 (C–Cl), 122.5, 125.7, 126.0, 128.8, 130.0, 131.2,
132.4, 133.8, 135.8, 136.6 (Aromatic), 164.0 (NHC=O),
198.8 (C=O); Anal. calcd for C15H10NO2Cl3: C % 52.59,
H % 2.94, N % 4.09; Found: C % 52.36, H % 2.86, N %
4.16.
IR KBr (cm-1) 3,277 (N–H), 3,064, 2,970 (C–H), 1,692
(C=O), 1,650 (NHC=O), 1,520 (NO2), 1,517 (C=C), 1,284
(C–N) and 630 (C–Br); 1H-NMR (CDCl3) d (ppm): 4.1 (s,
2H, –COCH2N), 7.15–7.65 (m, 7H, ArH), 11.3 (brs, 1H,
exchangeable, –NHCOCH2–); 13C-NMR (CDCl3)
d
(ppm): 30.2 (C–Br), 122.5, 125.5, 126.0, 126.8, 128.8,
131.8, 136.8, 138.8, 144.6, 148.0 (Aromatic), 16.6
(NHC=O), 192.8 (C=O); Anal. calcd for C15H10N2O4BrCl:
C % 45.31, H % 2.54, N % 7.05; Found: C % 45.36, H %
2.60, N % 7.16.
2-(chloroacetamido)-5,40-dichlorobenzophenone (4c)
General method for the syntheses of 2-{N,N-bis(20-
chloroethyl)}aminoacetamido-benzophenone
derivatives (5a–f)
IR KBr (cm-1) 3,283 (N–H), 3,065, 2,956 (C–H), 1,690
(C=O), 1,648(NHC=O), 1,514 (C = C), 1,288 (C–N) and
755 (C–Cl); 1H-NMR (CDCl3) d (ppm): 3.9 (s, 2H,
-COCH2N), 7.35–7.60 (m, 7H, ArH), 11.2 (brs, 1H,
exchangeable, -NHCOCH2–); 13C-NMR (CDCl3) d (ppm):
42.3 (C–Cl), 122.4, 125.9, 127.4, 129.3, 130.0, 131.2,
131.8, 133.6, 136.8, 138.0, 142.2 (Aromatic), 168.3
(NHC=O), 194.8 (C=O); Anal. calcd for C15H10NO2Cl3:
C % 52.59, H % 2.94, N % 4.09; Found: C % 52.48, H %
2.88, N % 4.04.
Method A A mixture of substituted 2-(bromo/chloro acet-
amido) benzophenone derivatives (4a–f) (10 mmol),
freshly distilled diethanolamine (30 mmol), anhydrous
sodium iodide (30 mmol) in dry DMF was heated for 20 h
at 120–130 °C on oil-bath. The reaction mixture was
treated with saturated aqueous sodium bicarbonate solu-
tion. The solution was extracted with ethyl acetate, washed
with brine and dried to get dihydroxy compound as oil
which without further purification was treated with excess
of phosphorous oxychloride (100 mmol) and heated under
reflux for 2.5–3 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
acetate (3 9 50 ml) and washed with 10 % sodium bicar-
bonate solution. The combined organic layer was dried
over anhydrous sodium sulfate, evaporated and passes
through silica column using ethyl acetate to give desired
final compounds (5a–f) as dark oil.
2-(bromoacetamido)-5-nitrobenzophenone (4d)
IR KBr (cm-1) 3,278 (N–H), 3,063, 2,968 (C–H), 1,692
(C=O), 1,655 (NHC=O), 1,518 (NO2), 1,514 (C=C), 1,288
(C–N) and 624 (C–Br); 1H-NMR (CDCl3) d (ppm): 4.2 (s,
2H, –COCH2N), 7.40–7.60(m, 8H, ArH), 9.80 (brs, 1H,
exchangeable, –NHCOCH2–); 13C-NMR (CDCl3)
d
(ppm): 30.6 (C–Br), 122.4, 125.4, 126.4, 127.8, 128.5,
130.6, 132.8, 140.0, 142.8, 148.0 (Aromatic), 162.0
(NHC=O), 195.8 (C=O); Anal. calcd for C15H11N2O4Br:
C % 49.61, H % 3.05, N % 7.71; Found: C % 49.68, H %
3.15, N % 7.64.
Method B A mixture of substituted 2-(bromo/chloro
acetamido) benzophenone derivatives (4a–f) (10 mmol),
bis(2-chloroethyl)amine hydrochloride (20 mmol), potas-
sium carbonate (20 mmol), sodium iodide (10 mmol), and
DMF was stirred for 48 h. The reaction mixture was
poured into excess of water and extracted with ethyl acetate
(3 9 50 ml). The recombined organic phases were dried
on sodium sulfate and evaporated in vacuo, and residue
2-(bromoacetamido)-20-chloro-5-nitrobenzophenone
(4e)
IR KBr (cm-1) 3,282 (N–H), 3,074, 2,973 (C–H), 1,694
(C=O), 1,648 (NHC=O), 1,522 (NO2), 1,522 (C=C), 1,270
123

Med Chem Res
was chromatographed on silica gel using ethyl acetate to
1,671 (NHC=O), 1,524 (NO2 assym stretch), 1,484 (C=C),
1,227, 1,123 (C–N), 759 (C–Cl); 1H-NMR (CDCl3) d
(ppm): 3.75(t, 4H, J = 7.84 Hz, –N(CH2CH2Cl)2) 4.12 (s,
2H, –COCH2N), 4.57 (t, 4H, J = 7.82 Hz, –N(CH2
CH2Cl)2), 7.50–7.68 (m, 8H, ArH), 11.26 (br s, 1H,
furnish compounds (5a–f).
2-{N,N-bis(20-chloroethyl)}aminoacetamido-5-
chlorobenzophenone (5a)
exchangeable, –NHCOCH2–); 13C-NMR (CDCl3)
d
Yield = 65 %, brown oil. Rf: 0.68 (chloroform: methanol:
9.5:0.5); IR KBr (cm21): 3,336 (N–H), 3,094, 2,943 (C–H),
1,699 (C=O), 1,685 (NHC=O), 1,486 (C=C), 1,214, 1,120,
1,097 (C–N), 754 (C–Cl); 1H-NMR (CDCl3) d (ppm): 3.05 (t,
4H, J = 8.0 Hz, –N(CH2CH2Cl)2), 3.42 (s, 2H, –COCH2N),
3.63 (t, 4H, J = 7.5 Hz, –N(CH2CH2Cl)2), 7.17–7.55(m, 8H,
ArH), 9.67 (br s, 1H, exchangeable, –NHCOCH2–); 13C-
NMR (CDCl3) d (ppm): 42.8 (2xC-Cl), 56.4 (2xC-N), 58.0
(CH2), 123.4, 125.7, 126.0, 128.4, 129.4, 130.4, 131.2, 132.8,
140.6, 142.8 (Aromatic), 174.0 (–NHC=O), 190.0 (C=O);
TOF MS ES1m/z: 437(M ? H ? Na)?.
(ppm): 43.2 (2xC-Cl), 56.4 (2xC-N), 58.2 (CH2), 122.4,
125.7, 126.8, 128.4, 130.5, 131.2, 132.8, 140.6, 142.8,
147.8 (Aromatic), 168.4 (–NHC=O), 195.0 (C=O); TOF
MS ES1 m/z: 447 (M ? Na)?.
2-{N,N-bis(20-chloroethyl)}aminoacetamido-20-chloro-
5-nitrobenzophenone (5e)
Yield = 52 %, red oil. Rf: 0.52 (ethyl acetate). IR KBr
(cm21): 3,350 (N–H), 3,080, 2,967 (C–H), 1,691 (C=O),
1,672 (NHC=O), 1,526 (NO2 assym stretch), 1,489 (C=C),
1,222, 1,124 (C–N), 758 (C–Cl); 1H-NMR (CDCl3) d
(ppm): 3.75 (t, 4H, J = 8.0 Hz, –N(CH2CH2Cl)2),
4.11 (s, 2H, –COCH2N), 4.55 (t, 4H, J = 8.1 Hz,
–N(CH2CH2Cl)2), 7.20–7.70 (m, 7H, ArH), 11.26 (br s,
1H, exchangeable, –NHCOCH2–); 13C-NMR (CDCl3) d
(ppm): 42.3 (2xC-Cl), 56.4 (2xC-N), 58.3 (CH2), 123.8,
125.7, 126.8, 127.2, 127.9, 128.4, 129.4, 130.4, 131.2,
134.8, 136.0, 152 (Aromatic), 168.0 (–NHC=O), 197.0
(C=O); TOF MS ES1 m/z: 481 (M ? Na)?.
2-{N,N-bis(20-chloroethyl)}aminoacetamido-5,20-
dichlorobenzophenone (5b)
Yield = 68 %, brown oil. Rf: 0.82 (chloroform: methanol:
9.8:0.2). IR KBr (cm21): 3,342 (N–H), 3,088, 2,940 (C–H),
1,694 (C=O), 1,680 (NHC=O), 1,476 (C=C), 1,219, 1,120,
1,086(C–N),756(C–Cl);1H-NMR (CDCl3) d(ppm):3.20(t,
4H, J = 7.6 Hz, –N(CH2CH2Cl)2), 3.70 (s, 2H, –COCH2N),
3.90 (t, 4H, J = 7.8 Hz, –N(CH2CH2Cl)2), 7.20–7.70(m, 7H,
ArH), 12.0 (br s, 1H, exchangeable, –NHCOCH2–); 13C–
NMR (CDCl3) d (ppm): 42.5(2xC-Cl), 56.8 (2xC-N), 58.5
(CH2), 123.2, 125.7, 126.8, 126.4, 127.9, 128.4, 129.4, 130.4,
131.2, 134.8, 140.8, 142.8 (Aromatic), 171.0 (–NHC=O),
197.0 (C=O); TOF MS ES1m/z: 472(M ? H ? Na)?.
2-{N,N-bis(20-chloroethyl)}aminoacetamido-40-chloro-
5-nitrobenzophenone (5f)
Yield = 56 %, red oil. Rf: 0.65 (ethyl acetate). IR KBr
(cm21): 3,354 (N–H), 3,016 (C–H), 1,685 (C=O), 1,677
(NHC=O), 1,521 (NO2 assym stretch), 1,485 (C=C), 1,219,
1,124 (C–N), 754 (C–Cl); 1H-NMR (CDCl3) d (ppm):
3.75(t, 4H, J = 7.84 Hz, –N(CH2CH2Cl)2), 4.12 (s, 2H,
–COCH2N), 4.57 (t, 4H, J = 7.82 Hz, –N(CH2CH2Cl)2),
7.50–7.70 (m, 7H, ArH), 11.38 (br s, 1H, exchangeable,
-NHCOCH2-); 13C-NMR (CDCl3) d (ppm): 42.4 (2xC-Cl),
55.6 (2xC-N), 57.5 (CH2), 122.8, 125.0, 125.8, 128.6,
130.2, 131.2, 137.0, 138.0, 144, 148 (Aromatic), 167.0
(–NHC=O), 195.0 (C=O); TOF MS ES1 m/z: 481
(M ? Na)?.
2-{N,N-bis(20-chloroethyl)}aminoacetamido-5,40-
dichlorobenzophenone (5c)
Yield = 64 %, brown oil. Rf: 0.91 (chloroform:methanol:
9.8:0.2). IR KBr (cm21): 3,345 (N–H), 3,087, 2,950 (C–H),
1,696 (C=O), 1,683 (NHC=O), 1,470 (C=C), 1,216, 1,122,
1,090 (C–N), 754 (C–Cl); 1H-NMR (CDCl3) d (ppm):
3.02(t, 4H, J = 8.2 Hz, –N(CH2CH2Cl)2), 3.45 (s, 2H,
–COCH2N), 3.64 (t, 4H, J = 8.0 Hz, –N(CH2CH2Cl)2),
7.20–8.58 (m, 7H, ArH), 11.27 (br s, 1H, exchangeable,
–NHCOCH2–); 13C-NMR (CDCl3) d (ppm): 42.5 (2xC-Cl),
56.8 (2xC-N), 58.5 (CH2), 123.2, 126.8, 128.4, 129.4, 130.4,
130.8, 131.2, 137.0, 138.0, 142.8 (Aromatic), 167.0
(–NHC=O), 194.0 (C=O); TOF MS ES1m/z: 448(M)?.
NBP alkylation assay
All compounds were checked for their NBP alkylating
activity by procedure mentioned before (Balazs et al.,
1970). 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 flask 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 flask was kept at 85 °C in a water bath for
2-{N,N-bis(20-chloroethyl)}aminoacetamido-5-
nitrobenzophenone (5d)
Yield = 54 %, red oil. Rf: 0.71 (ethyl acetate). IR KBr
(cm21): 3,329 (N–H), 3,091, 2,952 (C–H), 1,689 (C=O),
123

Med Chem Res
30 min. The solution was cooled immediately for 2–3 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 540 to 560 nm was recorded. This pro-
cedure was used by varying the heating time to obtain the
data tabulated in Table 3. The procedure was repeated
thrice for compound (5a–f).
cells only. For viability testing, 3-(4, 5-dimethylthiazole-2-
yl)-2, 5-diphenyl tetrazolium bromide (MTT) cell prolif-
eration assay was carried out (Mosmann, 1983). The cells
of all cell lines were plated out 24 h prior to testing in 96
well plates at a density of 3,000 cells/well in 100 lL of
the medium. After overnight incubation, triplicate wells
were treated with varying concentration of compounds
ranging from (1–100 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 MTT (5 mg/ml), and
cells were incubated for 3 h. The relative percentage of
metabolically active cells compared with untreated con-
trols and then determine on the basis of mitochondrial
conversion of MTT to formazan crystals which were
dissolved in dimethylsulfoxide (DMSO). Spectrophoto-
metric absorbance of sample was determined by micro-
plate reader (BIORAD) at 570–630 nm. Concentrations of
sample showing 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).
K values (i.e., rate constant) were calculated using the
formula:
K ¼ ðA2 ꢁ A1=T2 ꢁ T1Þ Where A = absorbance at
542–560 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-first-order rate constants for
NBP alkylation.
Half life of compound was calculated using the formula:
t1=2 ¼ 0:693=K
ADME prediction
ADME properties were calculated using Qikprop 2.5 tool
of Schrodinger software program designed by Professor
William L. Jorgensen. It evaluates the acceptability of
analogs based on Lipinski’s rule of five and Jorgensen’s
rule of three which are essential to insure drug-like phar-
macokinetic profile while using rational drug design. All
the analogs were neutralized before being used by Qikprop.
Â
% inhibition ¼ ðOD of controlÞ ꢁ ðOD of treatedÞ=
Ã
ðOD of controlÞ ꢂ 100:
Acknowledgments Authors are thankful to College Managing
Committee, SCOP, Nangal for providing necessary facilities to carry
out research work. Authors wish to express their gratitude to
Dr. Manoj Kumar, Professor of Pharmaceutical Chemistry, UIPS,
Panjab University, Chandigarh to carry out ADME study at his lab.
Authors are also thankful to ISFAL, ISF College of Pharmacy, Moga
(Punjab) for carrying out cell line studies and SAIF, Panjab Univer-
sity, Chandigarh for cooperation in getting the spectral data.
Antiproliferative assay in vitro
Cell culture
The following established in vitro human cancer cell lines
were applied: A 549 (Human lung cancer cells), COLO 205
(Human colon cancer cells), U 87 (Human primary glio-
blastoma cells), and IMR-32 (Human neuroblastoma cell
lines). All cell lines were obtained from NCCS Pune. All
chemical and solvent were purchased from Himedia. Cell
lines were maintained in desired media supplemented with
10 % inactivated fetal bovine serum, 100U/ml penicillin
and 100ul/ml streptomycin incubated at 37 °C and 5 %
CO2 in humidifier incubator. After attaining 80 % conflu-
ence, cells were subcultured by trypsinization with 0.25 %
trypsin solution under sterile conditions.
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