Nitric oxide release by N-(2-chloroethyl)-N-nitrosoureas: A rarely discussed mechanistic path towards their anticancer activity

Article abstract of DOI:10.1039/c4ra11137k

N-(2-Chloroethyl)-N-nitrosoureas (viz. carmustine (BCNU), lomustine) are used in critical cases of brain tumors or leukemia since these compounds are very toxic in nature. The mechanistic pathways of these compounds project the generation of isocyanates a

Full text of DOI:10.1039/c4ra11137k

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Nitric oxide release by N-(2-chloroethyl)-N-
nitrosoureas: a rarely discussed mechanistic path
Cite this: RSC Adv., 2015, 5, 2137
towards their anticancer activity†
Amrita Sarkar,‡ Subhendu Karmakar,‡ Sudipta Bhattacharyya, Kallol Purkait
*
and Arindam Mukherjee
N-(2-Chloroethyl)-N-nitrosoureas (viz. carmustine (BCNU), lomustine) are used in critical cases of brain
tumors or leukemia since these compounds are very toxic in nature. The mechanistic pathways of these
compounds project the generation of isocyanates along with [–N^N–CH2–CH2–Cl]+OHꢀ which, due
to its reactivity, will breakdown further to produce DNA adducts and various other metabolites. Almost
all of the literature predicting the mechanism does not discuss nitric oxide (NO) release as one of the
viable mechanistic pathways for these N-(2-chloroethyl)-N-nitrosoureas. Our studies with two aromatic
N-(2-chloroethyl)-N-nitrosoureas show that NO release may be a facile pathway for their activity. We
have probed the NO release through fluorescence and the traditional Griess reagent assay. The aqueous
stability studies show that the rates of decomposition of the two aromatic N-(2-chloroethyl)-N-
nitrosoureas are of the order of 10ꢀ2 minꢀ1. Initial studies of the cytotoxicity against two different cancer
cell lines show that they are quite efficient even under hypoxic conditions, compared to the clinical
compound BCNU, based on the results on human breast (MCF-7) and lung (A549) adeno carcinoma cell
lines. The IC50 values range from about 38–95 mM. Encouragingly the 1-(4-chloro-1,2-phenylene)bis(3-
(2-chloroethyl)-3-nitrosourea) (2a) is selectively more toxic to the A549 cancer cells (IC50 ¼ 41 ꢁ 4 mM)
than the normal mouse embryonic fibroblast (NIH3T3, IC50 63 ꢁ 4 mM) or non-tumorigenic human
embryonic kidney cell line (HEK293T, IC50 ¼ 83 ꢁ 2 mM) and arrests the cell cycle in the G2/M-phase in
the A549 cell line. Even under hypoxic conditions compound 2a is active at 49 ꢁ 5 mM.
Received 24th September 2014
Accepted 17th November 2014
DOI: 10.1039/c4ra11137k
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haematological toxicity.14–16 Following the evolution of N-nitro-
sourea drugs, it was found that from simple aliphatic, alicylic,
Introduction
aromatic or heterocyclic compounds the focus gradually shied
to more complex compounds conjugated with biorelevant
carrier moieties viz. amino acids, carbohydrates, nucleosides,
steroids etc. to achieve better toxicity along with specicity
towards cancer cells.3,7,17–27
N-(2-chloroethyl)-N-nitrosoureas
represent
a
class
of
compounds with a wide range of activities mainly used against
leukaemia and brain tumors.1–6 Carmustine and lomustine
(Scheme 1) are two such FDA approved drugs belonging to this
class and are mainly used in leukaemia and multiple
myeloma.7,8 Fotemustine and nimustine (Scheme 1) are newer
drugs in the same category under clinical trials.9–13 The varied
use of N-(2-chloroethyl)-N-nitrosoureas shows that although
they are of the same family, their pathway of action may be
different. All the clinically used nitrosourea drugs show
Department of Chemical Sciences, Indian Institute of Science Education & Research
Kolkata, Mohanpur, Nadia, Pin
– 741246, West Bengal, India. E-mail:
a.mukherjee@iiserkol.ac.in; Fax: +91-033-25873020; Tel: +91-033-25873031
† Electronic supplementary information (ESI) available: Figures showing aqueous
decomposition of compound 1–2a and nimustine hydrochloride in buffer (pH
ꢀ7.4, 6.0), tting plots for the determination of compounds 1a and 2a, cell
cycle analysis of 2a and nitric oxide release study of 1a, 2a and nimustine
hydrochloride, 1H and 13C NMR spectra of 1, 2, 1a and 2a. CCDC 1019082. For
ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/c4ra11137k
Scheme 1 Different nitrosoureas as chemotherapeutic agents.
RSC Adv., 2015, 5, 2137–2146 | 2137
‡ The authors pay equal contributions to this work.
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synthesized in one step using a isocyanate and the nitroso
derivatives were synthesized in a single step using NaNO2–
HCOOH.17,56 Hence, using two well dened synthetic steps we
can synthesize 1a and 2a from commercial precursors. The
characterization using NMR showed that the synthesized
compounds were pure. The aromatic protons of 1 and 1a are
multiplets instead of doublets (details in Experimental section).
This may be assigned to the difference in spatial orientation of
the N-(2-chloroethyl)-N-nitrosourea moieties reducing the
symmetry of the molecule as found in the crystal structure of 1a.
The analytical purity of the bulk products were further
conrmed by CHN analysis. The ESI-MS of the compounds are
also in good agreement with proposed molecular formulation.
Scheme 2 Representative scheme for synthesis of nitrosoureas (1a,
2a) (where 1, 2 and 1a were synthesized according to literature
procedure17,56). (i) 2-Chloroethyl isocyanate (2 equiv.), DCM/THF, 0 ꢂC,
0 ꢂC to RT, 6 h (ii) NaNO2–HCO2H, 0–5 ꢂC, 4 h.
Although nitrosourea contain a nitric oxide (NO) group, the
dissociation pathways shown mostly does not include the
proposition of NO release so far except for the work of Lown
et al. which although does not show any direct evidence of NO
release but through detection of other intermediates has dis-
cussed NO release as one of the pathways.28–30 The mechanism
of action of nitrosoureas are known to be complex and various
species are postulated on direct or indirect evidences.4,28,29,31–34
Most of the mechanisms proposed stresses mainly upon the
formation of isocyanates, [–N^N–CH2–CH2–Cl]+OHꢀ and
+CH2–CH2–Cl in addition to various other minor species but no
NO release is generally reported except in two cases28,30 where
the authors argued in favour of NO release as one of the path-
ways. Except for those two isolated reports the other
studies support the formation of HO–N]N–CH2–CH2–Cl, and
+CH2–CH2–Cl intermediates.2,31,33,35–38
Structural description
The compound 1a (Fig. 1) was characterized by single crystal X-
ray crystallography. It crystallizes in triclinic space group P1.
ꢀ
Single crystals suitable for X-ray crystallography were grown by
keeping a saturated DMSO solution at 20 ꢂC for a day. The
important crystallographic parameters and selected bond
length and angles have been summarised in Tables S1 and S2
(ESI†) respectively. The molecular structure of 1a revealed that
C–N bonds in urea group are asymmetric. The C(7)–N(4) adja-
˚
cent to phenyl ring has a bond distance of 1.340(3) A whereas
the same for C(7)–N(5), adjacent to the nitroso group, is 1.424(3)
˚
A showing the inuence of the phenyl ring on the double bond
˚
˚
character. The N–O bond lengths of 1.223(2) A and 1.224(3) A
are in good agreement with earlier reported N-nitrosourea
monomer.57,58 The molecule has a intramolecular hydrogen
˚
Our studies presented here show that NO release seems to be
a facile and viable mechanistic pathway for aromatic N-(2-
chloroethyl)-N-nitrosoureas. Ever since the mechanism of
endogenous nitric oxide (NO) was established in 1982 it has been
extensively probed for various biomedical applications.39–51 It is
known that NO at higher doses (exceeding 1 mM) trigger cell cycle
arrest52 thus NO donors may be potent anticancer agents.42,53–55
We have synthesized two aromatic N-(2-chloroethyl)-N-
nitrosourea (Scheme 2) to study their stability prole, NO
release ability and attempt to correlate the aqueous decompo-
sition and NO release ability to their cytotoxic activity towards
cancer cells. Our target compounds were 1a and 2a out of which
the synthetic procedure for 1a was reported earlier but the
compound was not studied for aqueous stability, NO release or
in vitro cytotoxicity.17,56 1 and 2 were good precursors to perform
comparative studies with their nitroso derivatives, 1a and 2a
whenever needed.
bonding (2.729 A) between O(3) and N(1) and intermolecular
hydrogen bonding between O(1) of one molecule to N(4) of a
neighbouring molecule.
Stability studies
The half lives of the N-nitrosoureas were monitored via UV-vis
absorption spectroscopy in buffer at two different pH, 7.4 and
6.0. Tumour cells have hypoxic regions which may have rela-
tively lower interstitial pH due to the accumulation of metabolic
products from glycosis.59,60 Since the decomposition may be pH
Results and discussion
Syntheses and characterization
The synthesis of ureas (1, 2), nitrosoureas (1a, 2a) proceeded as
depicted in Scheme 2. 1,10-(1,2-phenylene)bis(3-(2-chloroethyl)
urea) (1), 1-(4-chloro-1,2-phenylene)bis(3-(2-chloroethyl)urea)
(2) and 1,10-(1,2-phenylene)bis(3-(2-chloroethyl)-3-nitrosourea)
(1a) were prepared using earlier reported literature proce-
Fig. 1 Molecular structure of 1a with 50% probability of thermal
dure.17,56 The aromatic 2-chloroethylurea derivatives were ellipsoids. Hydrogen atoms have been omitted for clarity.
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Table 1 Aqueous decomposition rate (kD) and t1/2 of compounds 1a,
2a and nimustine hydrochloride in phosphate buffer medium (pH ¼ 7.4
and 6.0)
dependent, more decomposition at lower pH may lead to the
compound being degraded faster which may prohibit its entry
inside a cell. Hence the compounds were studied for their
decomposition at pH 6.0. The decrease in the absorption
maxima around 196–242 nm of the compounds (1a, 2a and
nimustine hydrochloride) were monitored with time. The rate
kD (minꢀ1
a
)
t1/2 (min)
pH
7.4
pH
of aqueous decomposition (kD) and corresponding half life (t1/2
)
Compounds
6.0
7.4
6.0
were calculated by plotting the ln(Abs) values vs. time plot. It
was observed from the aqueous decomposition rate for both 1a
and 2a that the rate of 1a is comparatively faster than 2a. Both
1a and 2a were found to be decomposing faster than nimustine
hydrochloride (Fig. 2). In lower pH (pH ¼ 6.0) in every case there
is a decrease in aqueous decomposition rate although the
difference is less (Table 1). The absorbance vs. wavelength plots
were shown in Fig. S1–S3 (ESI†) and the inset gures show
corresponding ln(Abs) vs. time plot. The nitrosoureas 1a and 2a
are reactive due to the presence of the NO group. The rate of
decomposition at pH 7.4 shows the order is 1a > 2a (Table 1). 1a
decompose faster than 2a and the only difference in the
formulation of 2a is the presence of the chloroaryl moiety
instead of aryl in 1a (Scheme 2). Hence the +R effect of the
chloro group is responsible for slowing the aqueous decompo-
sition rate.
1a
2a
5.61(3) ꢃ 10ꢀ2
1.76(9) ꢃ 10ꢀ2
3.85(4) ꢃ 10ꢀ3
1.72(9) ꢃ 10ꢀ2
1.20(4) ꢃ 10ꢀ2
9.73(3) ꢃ 10ꢀ4
12
39
180
40
58
712
Nimustine HCl
a
The value in parentheses shows standard error. Experiments were
carried out in triplicates.
is ca. 2.81 ꢃ 10ꢀ2 for 1a and 8.80 ꢃ 10ꢀ3 minꢀ1 for 2a. We found
that at pH 6.0 the rate of decomposition slows down and the
rate is 1.72(9) ꢃ 10ꢀ2, 1.20(4) ꢃ 10ꢀ2 and 9.73(3) ꢃ 10ꢀ4 minꢀ1
for 1a, 2a and nimustine respectively (Table 1). Hence, it shows
that when the pH is acidic (pH 6.0), the reduction in decom-
position rate is more pronounced for nimustine as compared to
1a and 2a. In fact the decomposition rate for compound 2a is
similar in both hypoxia and normoxia. Hence, the activity of 2a
in hypoxia and normoxia may not show much difference. 1a and
2a have two dissociable N-(2-chloroethyl)-N-nitrosourea moie-
ties per molecule which is also more atom economic compared
to nimustine since there would be dissociation of one arene by
product per two alkylating moiety and NO released. One would
expect that these aqueous decomposition data would show
correlation with the anticancer activity.
The UV spectrum also shows multiple isobestic point
(Fig. S1–S3, ESI†) indicating that there are two different species
in solution, which are UV active and one is forming steadily
from the other. From an apparent look it would appear that
nimustine which is
a N-(2-chloroethylnitrosourea) class
compound and is in clinical trial has decomposition rate much
slower than that of 1a or 2a (Table 1). However, it should be
noted that per molecule there are two N-(2-chloroethyl)-N-
nitrosourea moieties in 1a and 2a in contrast to one in nimus-
tine. Accounting for this fact it shows that the aqueous
decomposition rate per N-(2-chloroethyl)-N-nitrosourea moiety
Potency to inhibit cancer cells in vitro
All the four compounds were probed in vitro against human
breast adenocarcinoma cell line (MCF-7), human lung adeno-
carcinoma epithelial cell line (A549), non tumerogenic human
embryonic kidney cell (HEK293T) and mouse embryonic bro-
blast cell line (NIH3T3) by MTT assay and cisplatin was used as
a standard to check the quality of data in each 96 well plate. Two
well known compounds belonging to N-nitrosourea family
BCNU (FDA approved drug) and nimustine (under clinical
trials) were also tested to evaluate their corresponding IC50
values under same conditions in MCF-7 and A549, HEK293T
and NIH3T3 cell lines. The data are summarized in Table 2 and
corresponding IC50 plots of 1a and 2a in different cancer cell
lines using GraphPad Prism 5® are given in Fig. S4–S9, (ESI†).
The hypoxic activation was also probed by cytotoxicity assay of
1a and 2a in hypoxic conditions using oxygen percentage of
1.5%. Both the nitrosourea compounds are more active in A549
as compared to MCF-7 (Table 2). Compound 1a and 2a have
similar cytotoxicity (IC50 ¼ 38 ꢁ 1 mM for 1a and 41 ꢁ 5 for 2a) in
A549 under normoxic condition. 1a bearing the N-(2-chloro-
ethyl)-N-nitrosourea without the chloro in the aryl moiety, has
the fastest rate of decomposition and showed better activity
(Table 2). In both A549 and MCF-7 cell line 1a and 2a show
similar activity in normoxia and hypoxia (Table 2). The MTT
Fig. 2 Decrease in absorbance (normalised) vs. time plot for aqueous
stability of compounds 1a, 2a and nimustine HCl in phosphate buffer
(10 mM) of pH 7.4 and 6.0. The decrease in absorbance at l ¼ 236 nm
for 1a, 242 nm for 2a, and 232 nm for nimustine HCl with time has been
monitored and plotted.
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Table 2 Cytotoxicity data of compounds 1–2a in MCF-7, A549, HEK293T and NIH3T3 in normoxia (18% O2) and hypoxia (1.5% O2)
a
IC50 (mM) ꢁ SD
HEK293T
Normoxia
NIH3T3
MCF-7
A549
Compounds
Normoxia
Hypoxia
Normoxia
Hypoxia
Normoxia
1
>500
n.dc
>500
n.d
n.d
n.d
2
1a
2a
BCNU
Nimustine HCl
Cisplatin
244 ꢁ 1
95 ꢁ 2
90 ꢁ 3
>150b
n.d
>350
n.d
n.d
n.d
89 ꢁ 2
86 ꢁ 1
>150b
>150b
19 ꢁ 1
38 ꢁ 3
41 ꢁ 4
>150b
>150b
22 ꢁ 2
40 ꢁ 2
49 ꢁ 5
>150b
>150b
24 ꢁ 2
86 ꢁ 2
83 ꢁ 2
>150b
>150b
9 ꢁ 2
39 ꢁ 3
63 ꢁ 4
>150b
>150b
6 ꢁ 2
>150b
14 ꢁ 1
a
IC50 ¼ concentration required to effect 50% inhibition of cell growth, values are means ꢁ standard deviation of three independent experiments
carried out by triplicates, p < 0.05. IC50 values were calculated by variable slope model using GraphPad Prism 5®. Cells (6 ꢃ 103 per well) were treated
b
c
with increasing concentrations of compounds for 48 h. At 150 mM survival rate was 75–80%. n.d ¼ not determined.
assay of BCNU (a clinical drug) and nimustine under same
conditions as our compounds (details in Experimental section)
does not show any signicant killing (survival > 75%) up to 150
mM in normoxia or hypoxia. We also tested 1a and 2a in a non
tumerogenic (HEK293T) and a primary cell line (NIH3T3).
Primary cell lines are generally difficult to culture in vitro
conditions yet the IC50 values suggest that 2a is much less toxic
to the HEK293T and NIH3T3 as compared to its activity in A549
cell line. 1a was less toxic for HEK293T but showed similar
toxicity in NIH3T3 when compared with A549.
We also performed some initial studies on the cell cycle
inhibition by ow cytometry (FACS) with 2a. Aer 24 hours of
incubation with 2a the respective DNA content were analysed by
FACS (Table S3†). The results showed that compound 2a lead to
arrest of A549 cells in G2/M phase (Fig. 3 and S10, ESI†) signi-
fying that those cells where there has been signicant DNA
damage could not enter the next phase M, of the cell cycle, and
if somehow they have escaped the G2 phase arrest, then the
cells were trapped in M phase. The optical microscopy image
data of A549 cells aer 24 h treatment of 2a also support the cell
cycle arrest data as visible change in nuclear morphology
(nuclear elongation) was observed (Fig. S11†).
Nitric oxide release studies
In order to monitor if release of nitric oxide (NO) is possible
from the N-(2-chloroethyl)-N-nitrosoureas 1a and 2a Griess
reagent test was performed (it should be noted that Griess
reagent detects NO2ꢀ which is formed from NO by reaction with
oxygen).52,61 The NO release reactions were performed by dis-
solving 1a, 2a and nimustine in a sealed vial in DMSO–water
(7 : 3 v/v) and aer 5 min injecting the head space gas in to the
Griess reagent vial. The nitrite detected was the oxidised
product of nitric oxide in the reaction with Griess reagent
(Scheme S1†) producing increase of absorbance at ꢄ540 nm.
The test was found to be positive by development of the purple
coloration. This was done over a period of time and the purple
colouration was found to increase conrming the release of NO
(Fig. 4). Since same amount of overhead gas from each sample
was used the data obtained may show the trend in NO release
although it would not be able to quantitate. The amount of NO
released is of the order nimustine > 1a > 2a. Later we performed
the same experiment using the traditional solution phase
test52,61 (Fig. S12 & S13, ESI†) and found the same trend as
obtained using the overhead gas. The rate constants (kNO) were
calculated from the slope of ln(Abs) vs. time plots (Fig. S14,
ESI†) and the rate constant (kNO) falls in the order, nimustine >
1a > 2a. Release of labile NO was conrmed and estimated by
the increase of absorbance produced by the resultant dye at
ꢄ540 nm. The estimated NO release from 1a, 2a and nimustine
hydrochloride were compared with the data obtained from a
well known nitrite donor compound, sodium nitrite, under the
same experimental condition. Both 1a and 2a took ca. 4 h for
achieving saturation but for nimustine it took much less time
ca. 132 min (Fig. S12, ESI†).
However, it should be noted that the solution phase (Table 3)
NO release rates were under highly acidic conditions in solution
(see Experimental section for details) which is different from
normal physiology or cancer cells. Hence, the quantitation of
NO release is only indicative and performed to compare the
maximum NO release ability of 1a and 2a with nimustine.
Fig. 3 Cell cycle analysis of A549 cells after 24 h exposer to 2a in three
different concentrations 15 mM, 25 mM and 30 mM respectively.
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Fig. 4 Change in absorbance vs. wavelength (lmax ¼ 540 nm) when headspace gas from (A) 1a and (B) 2a and (C) nimustine HCl giving positive
Griess reagent test.
Table 3 Estimation of NO production by 1a, 2a and nimustine HCl by Griess reagent testa
Compounds
[NO]t (mM)
kNO (minꢀ1
)
t1/2 (min)
td (min)
NO released (mol%)
1a
2a
25.5
23.5
42.9
5.88 ꢃ 10ꢀ3
4.19 ꢃ 10ꢀ3
1.48 ꢃ 10ꢀ2
118
165
47
230
234
132
20.4
18.8
68.6
Nimustine HCl
a
Compound concentration (1a, 2a and nimustine HCl) ¼ 62.5 mM. [NO]t ¼ total NO release, kNO ¼ rate of NO release, t1/2 ¼ half life of NO release,
td ¼ duration of NO release. All the above data are mean of two independent experiments.
Compound 1 and 2 gave no colouration in Griess reagent assay triazole DAA-Tz (Fig. 5D). We have also compared the uores-
under the experimental conditions indicating that there may cence of DAA-Tz formed by a known nitrosating agent NOBF4
not be an alternate reactive species that can mislead the Griess which further supports that nitric oxide is releasing from N-(2-
test. The NO release by nimustine or other aromatic N-(2- chloroethyl)-N-nitrosourea class of compounds studied here.
chloroethyl)-N-nitrosoureas viz. 1a, 2a, is not known as per the
Our investigation to probe the NO release by probing the
literature. Out of the numerous literatures on detailed mecha- overhead gas as well as the solution of 1a and 2a using the
nistic pathways2,31,33,36–38 of the N-(2-chloroethyl)-N-nitrosoureas traditional Griess reagent assay and by uorescence spectros-
only the work of Lown et al.28–30 indicates NO release by BCNU, copy forming DAA-triazole showed that NO indeed gets released
MeCCNU and CCNU as one of the possible pathways based on from these compounds (Fig. 4, 5 and Table 3). Based on the
the intermediates formed and detected, due to NO release. above results we have proposed an alternate mechanism to
However, no NO release experiment was reported. Our results show the nitric oxide release in Scheme 3. Apart from the
also strongly agree with the NO release pathway and we have evidence of NO release the mechanistic proposal was based on
direct evidence of NO release by using the overhead gas for 1a, the mass spectrometry of solutions of 1a performed in 2 : 1 v/v
2a and nimustine in Griess reagent assay.
MeCN–H2O mixture (2a being similar to 1a only the latter was
The detection of released nitric oxide from N-(2-chloroethyl)- studied through ESI-MS). We found that ESI-MS data shows the
N-nitrosourea (1a) as well as nimustine was also conrmed by presence of imidazolone and isocyanates as the major products
uorescence studies. Among various uorescent probes avail- (Fig. S17A & S17B, ESI†) of aqueous decomposition. We also
able aromatic ortho-diamino compounds are most commonly found existence of oxadiazolium cation in the ESI-MS of 1a
used.62–64 1,2-Diamino anthraquinone (DAA) may be used as a (Scheme 3, Fig. S17A & S17B, ESI†). Hence we proposed the
sensitive and specic NO probe.65 1,2-Diamino compounds react mechanistic pathway as depicted in Scheme 3, where path A
with nitric oxide (NO) in presence of O2 to produce a uorescent shows the formation of oxadiazolium cation.
triazole derivative (DAA-Tz) (Scheme S2, ESI†) with a new emis-
The proposed pathways show that the major end products
sion maxima.65 The emission spectra of DAA in DMSO (50 mM) may be similar whether NO is released or not. We propose that
exhibit peak maxima at 638 nm with a weak shoulder at 570 nm NO release can happen at different stages depending on the
(Fig. 5A). Upon treatment 50 mM of 1a the solution exhibits a new compound and pathway followed (Scheme 3, Fig. S17A & S17B,
band at 562 nm overlapping with a band at 640 nm showing that ESI†). The reason behind this proposition is that the aqueous
both DAA and DAA-Tz exist in the solution (Fig. 5B). When we decomposition and NO release are not the same as found
added the headspace gas (20 mL) generated from compound 1a through our results. 1a and 2a show a higher decomposition
the formation of triazole derivative (DAA-Tz) was conrmed by rates and higher cytotoxicity but their NO release rate is slower.
the peak at 574 nm (Fig. 5C). For nimustine hydrochloride the Nimustine on the other hand shows a slower decomposition
peak appeared at 571 nm was the conrmation of formation of rate but the NO release rate is higher even at pH 7.4 as per the
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in BCNU the ESI-MS data shows evidence of the aziridinium
cation (Scheme S4†) which was not found in 1a or nimustine.
Hence, for BCNU path C is feasible. It should be noted that the
path C cannot be totally excluded for 1a or nimustine since the
stability of the aziridinium formed under the conditions of ESI-
MS would dictate its detection. The results bring more clarity to
why these N-(2-chloroethyl)-N-nitrosoureas are active against
different type of cancer instead of all of them being selective to
one particular type of cancer. One strong reason could be that
they have different mechanistic pathway.
The amount of NO released per mole of compound would
inuence the cytotoxicity. 1a and 2a shows good cytotoxicity
under the in vitro testing conditions although the NO release rate
is higher for nimustine. The reason may be that the NO is
released before the compound can enter the cell making it less
active. The clinically approved BCNU as per literature has a half
life of 15–20 min in plasma66 but 2a has a half-life of 39 min in
buffer and shows better anticancer activity than BCNU on MCF-7
and A549. Apart from the compounds being more effective in the
probed cell lines compared to BCNU and nimustine, the most
important factor here is the NO release by the two aromatic N-(2-
chloroethyl)-N-nitrosoureas and nimustine at physiologically
relevant pH 6.0–7.4. The results of NO release studies conrm
that nitric oxide release is happening at physiological pH and
hence may inuence the mechanism of action for the aromatic
N-(2-chloroethyl)-N-nitrosoureas similar to that highlighted for
aliphatic acyclic N-(2-chloroethyl)-N-nitrosoureas by the earlier
work28,30 of Lown et al. We found that faster NO release, may not
lead to better activity. The compound may be deactivated before
it can reach the target if the NO release is faster. It should be
borne in mind that NO release, is not the sole pathway of action
for N-(2-chloroethyl)-N-nitrosoureas and our results also support
the same. However, NO release is happening in all the
compounds. More work has to be done, keeping the NO release
pathway in mind so that we can gain more insight about the
correlation of NO release rate and cytotoxicity.
Fig. 5 (A) DAA (50 mM in DMSO): normalized emission spectrum, lex
¼
490 nm. (B) DAA (50 mM in DMSO) + 1a (50 mM in DMSO): normalized
emission spectrum, lex ¼ 490 nm. (C) DAA (50 mM in DMSO) +
headspace gas of 1a (20 mL): normalized emission spectrum, lex
¼
490 nm. (D) DAA (50 mM in DMSO) + nimustine HCl (50 mM in DMSO):
normalized emission spectrum, lex ¼ 490 nm.
Scheme 3 Schematic representation of proposed mechanistic path-
ways of N-nitrosourea (1a).
Experimental
Materials and methods
All chemicals and solvents were purchased from commercial
sources. Solvents were distilled and dried prior to use. UV-
visible measurements were done using Perkin Elmer
lambda 35 spectrophotometer. FT-IR spectra were recorded
using Perkin-Elmer 120-000A in KBr pellets. Fluorescence
measurements were carried out in Horiba Jobin Yvon Fluo-
rolog instrument. The excitation source used here is a steady
overhead gas studies. However, Nimustine shows a poor in vitro
cytotoxicity prole. Hence, it appears that the rate of decom-
position does not provide indication of released NO. It appears
that if NO is released at a faster rate than the cytotoxicity of the
compound becomes poor which may be due to poor perme-
ability of the resultant compound. Our ESI-MS studies of
decomposition of BCNU and nimustine supports that the
dissociation pathways are different (Schemes S3, S4 and
Fig. S18, S19†). In case of 1a based on the NO detection and the
ESI-MS data only path A and B are followed (Scheme 3). Based
on the differences in the data it can be said that the NO release
do not happen through the same pathway in nimustine and 1a
or 2a. In case of nimustine the percentage of path B may be
higher than path A since more NO is released in nimustine and
we do not nd the oxadiazolium cation as found in 1a. However,
1
state 450 W Xe lamp. H & 13C NMR spectra were measured
using Bruker Avance III 500 MHz spectrometer at room
temperature. The chemical shis are reported in parts per
million (ppm). The proton decoupled 13C NMR spectra are
reported. Elemental analyses were performed on a Perki-
nElmerEA 2400 CHNS series elemental analyzer. Electro-
spray ionization mass spectra were recorded using micro-
mass Q-Tof microTM, Waters by +ve mode electrospray
ionization. The synthetic yields reported are of isolated
analytically pure compounds.
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(CONH), 151.6 (CONH), 132.8 (ArC), 131.2(ArC), 128.1 (ArC),
127.3 (ArC), 127.0 (ArC), 125.6 (ArC), 40.2 (CH2Cl), 38.9
(CH2NNO); FT-IR (KBr, cmꢀ1): 3286 (br), 2963 (w), 1609 (s), 1576
(s), 1484 (s), 1452 (s), 1436 (m), 1370 (s), 1249 (s), 1192 (w), 1127
(w), 1066 (w), 946 (w), 752 (s), 656 (s); elemental analysis calcd
for C12H13Cl3N6O4: C 45.01, H 3.18, N 20.42, found: C 44.76, H
3.31, N 20.24; ESI-MS m/z 433.00 [M + Na]+, found 433.00.
Syntheses
1,10-(1,2-Phenylene)bis(3-(2-chloroethyl)urea (1).
1
was
prepared following a previously reported method.56 Yield 97%.
Mp 167 ꢂC; 1H NMR (500 MHz, DMSO-d6) (Fig. S20†): d 7.98 (s,
2H, ArNH), 7.50 (m, 2H, ArH), 6.99 (m, 2H, ArH), 6.85 (t, J ¼ 5.5
Hz, 2H, NHCH2), 3.66 (t, J ¼ 6.25 Hz, 4H, CH2Cl), 3.42 (m, 4H,
NHCH2). 13C NMR (125 MHz, DMSO-d6) (Fig. S21†): d 155.7
(CONH), 131.3 (ArC), 123.5 (ArC), 123.3 (ArC), 44.2 (CH2Cl), 41.4
(CH2NH); FT-IR (KBr, cmꢀ1): 3286 (br), 2963 (w), 1609 (s), 1576
(s), 1484 (s), 1452 (s), 1436 (m), 1370 (s), 1249 (s), 1192 (w), 1127
(w), 1066 (w), 946 (w), 752 (s), 656 (s); elemental analysis calcd
for C12H16Cl2N4O2: C 45.15, H 5.05, N 17.55, found: C 44.70, H
5.08, N 17.23; ESI-MS m/z 341.05 [M + Na]+, found 341.06 [M +
Na]+.
X-ray crystallography
Single crystal of 1a was mounted using loops on the goniometer
head of a SuperNova, Dual, Cu at zero, EOS diffractometer. The
crystal was kept at 100.00(10) K temperature during data
collection. Using Olex2,67 the structure was solved with the
Superip68 structure solution program using Charge Flipping
and rened with the ShelXL69 renement package using Least
Squares minimisation.
1-(4-Chloro-1,2-phenylene)bis(3-(2-chloroethyl)urea) (2).
2
was synthesized through a known literature procedure.56 Yield
90%. Mp: 169–172 ꢂC (dec); 1H NMR (500 MHz, DMSO-d6)
(Fig. S24†): d 8.12 (s, 1H, ArNH), 7.99 (s, 1H, ArNH), 7.76 (d, J ¼
2.5 Hz, 1H, ArH), 7.43 (d, J ¼ 8.5 Hz, 1H, ArH), 7.06 (t, J ¼ 5.5 Hz,
1H, NHCH2), 7.00 (dd, J1 ¼ 8.5 Hz, J2 ¼ 2.5 Hz, 1H, ArH), 6.77 (t, J
¼ 5.75 Hz, 1H, NHCH2), 3.67 (m, 4H, CH2Cl), 3.43 (m, 4H,
CH2NH); 13C NMR (125 MHz, DMSO-d6) (Fig. S25†): d 155.7
(CONH), 155.2 (CONH), 133.7 (ArC), 128.8 (ArC), 127.5 (ArC),
125.4 (ArC), 122.2 (ArC), 121.5 (ArC), 44.2 (CH2Cl), 44.1 (CH2Cl),
41.5 (CH2NH), 41.4 (CH2NH); FT-IR (KBr, cmꢀ1): 3332 (s), 1663
(s), 1592 (s), 1558 (s), 1478 (m), 1411 (m), 1370 (m), 1370 (s),
1249 (s), 1192 (w), 1127 (w), 1066 (w), 946 (w), 752 (s), 656 (s);
elemental analysis calcd for C12H15Cl3N4O2: C 40.76, H 4.28, N
15.85, found: C 40.46, H 4.11, N 15.48; ESI-MS m/z 353.03 [M +
H]+, found 353.26.
Aqueous stability studies
The determination of the half life of the nitrosoureas was
carried out by monitoring the decrease of absorbance in UV-vis
spectrophotometer.32 5 mL (1 ꢃ 10ꢀ2 M) of acetonitrile stock
solution of nitrosoureas were taken in 995 mL phosphate buffer
(50 mM, pH ¼ 7.4). Absorbances were recorded at 5 min time
interval at 298 K. The aqueous decomposition rate constant
were determined by plotting ln(Abs) vs. time data where from,
the slope, rate of aqueous decomposition (kD) and corre-
sponding half life (t1/2) were determined.
Nitric oxide (NO) release studies
Griess reagent assay
1,10-(1,2-Phenylene)bis(3-(2-chloroethyl)-3-nitrosourea) (1a).
It was prepared following the previous reported literature
method.56 Yield 85%. Mp: 94 ꢂC; 1H NMR (500 MHz, CDCl3)
(Fig. S28†): d 9.16 (s, 2H, ArNH), 7.65 (m, 2H, ArH), 7.37 (m, 2H,
ArH), 4.23 (t, J ¼ 6.5 Hz, 4H, CH2Cl), 3.54 (t, J ¼ 6.5 Hz, 4H,
CH2NNO); 13C NMR (125 MHz, CDCl3) (Fig. S29†): d 151.7
(CONH), 129.8 (ArC), 127.4 (ArC), 125.9 (ArC), 40.1 (CH2Cl), 38.8
(CH2NNO); FT-IR (KBr, cmꢀ1): 3286 (br), 2963 (w), 1609 (s), 1576
(s), 1484 (s), 1452 (s), 1436 (m), 1370 (s), 1249 (s), 1192 (w), 1127
(w), 1066 (w), 946 (w), 752 (s), 656 (s); elemental analysis calcd
for C12H14Cl2N6O4: C 38.21, H 3.74, N 18.80, found: C 38.45, H
3.91, N 18.23; ESI-MS m/z 399.03 [M + Na]+, found 399.04.
1-(4-Chloro-1,2-phenylene)bis(3-(2-chloroethyl)-3-nitrosourea)
(2a). In a round bottom ask, 2 (0.31 g, 1.00 mmol) was taken in
98% formic acid and kept in ice-salt bath. Aer cooling solid
NaNO2 (0.83 g, 12.00 mmol) was added to it in small portions
over 1 h duration. It was kept in 0–5 ꢂC for 4 h. A greenish
precipitate began to appear. Aer completion of reaction the
mixture was poured on crushed ice and kept in freezer for 1 h
for complete precipitation. The precipitate was ltered washed
with diethyl ether for several times and dried for P2O5 for
overnight. Yield 83%. Mp: 97–102 ꢂC (dec); 1H NMR (500 MHz,
CDCl3) (Fig. S32†): d 9.19 (s, 1H, ArNH), 9.06 (s, 1H, ArNH), 7.76
(d, J ¼ 2.5 Hz, 1H, ArH), 7.57 (d, J ¼ 8.5 Hz, 1H, ArH), 7.34 (dd, J1
¼ 8.5 Hz, J2 ¼ 2.5 Hz, 1H, ArH), 4.23 (m, 4H, CH2Cl), 3.54 (m,
CH2NNO); 13C NMR (125 MHz, CDCl3) (Fig. S33†): d 151.8
Headspace gas (NO) detection by Griess reagent. The NO release
experiments were carried out using the headspace gas gener-
ated in the sealed vial of the DMSO–buffer solution (7 : 3) of 1a,
2a and nimustine hydrochloride. The overhead gas were
syringed out in a gas tight syringe (2 ꢃ 2 mL) and purged into a
solution containing 200 mL freshly prepared Griess reagent
(0.1% sulfanilide, 1% N-naphthylethylenediamine dihydro-
chloride and 1% phosphoric acid) and 600 mL 10 mM phosphate
buffer (pH ¼ 7.0). The increase in absorbance at 540 nm was
measured with time. However, the reaction of NO with Griess
reagent is slower and hence some amount of NO escapes the
detection.
Solution phase Griess test. The NO release from 1a and 2a was
conrmed by the estimation of nitrite using Griess reagent. To
600 mL of 10 mM phosphate buffer (pH ¼ 7.0), 195 mL freshly
prepared Griess reagent (0.1% sulfanilide, 1% N-naphthyl-
ethylenediamine dihydrochloride and 1% phosphoric acid) was
added followed by the addition of compound solution (5 mL) of
1a and 2a in DMSO (1 ꢃ 10ꢀ2 M). The estimation of nitric oxide
or nitrite ion was measured by observing the absorbance at
540 nm which continuously increased over the time period of
the experiment. The data were recorded every 6 min time
interval. Standard calibration curve was derived from 2–60 mM
sodium nitrite standard solutions.
Fluorescence spectroscopy. Fluorescence of 50 mM 1,2-dia-
minoanthraquinone (DAA) solution in DMSO was recorded
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using lex ¼ 490 nm. 8 mL of 5 mM DMSO solution of 1a (nal and nimustine were solubilised in acidic ethanol followed by
conc. 50 mM) in 50 mM DAA solution was added and spectra dilution with water.
recorded using lex ¼ 490 nm. Same method was followed for
For hypoxic condition the O2% of the CO2 incubator (ESCO
nimustine hydrochloride. To detect nitric oxide in the head- cell culture CO2 incubator, model: CCL-170T-8-UV) was main-
space gas evolved from 1a, a 20 mM DMSO solution of 1a was tained at 1.5%. Even for incubation under hypoxia 48 h period
prepared in a sealed vial and 20 mL headspace gas was syringed was used. The drug additions were all done in normoxic
out in a gas tight syringe and purged into the 50 mM aerated DAA condition and the 96 well plates were then placed in incubator.
solution and aer 10 min the spectra was recorded using lex
490 nm.
¼
It took ca. 30 min for the incubator to reach oxygen percentage
at 1.5%.
pH and conductivity study. The NO release would lead to
Aer 48 h the drug containing media was removed and fresh
change in pH due to formation of nitrous/nitric acid hence the media was added to each well and successively treated with
change in pH was monitored using an aqueous solution con- 20 mL of a 1 mg mLꢀ1 MTT in 1ꢃ PBS (pH ¼ 7.2). Aer 3 h of
taining 20% DMSO and 1a. The pH decrease over time was incubation, media was removed and 200 mL of DMSO were
recorded using
(Fig. S15†).
a
Mettler Toledo SevenEasy pH meter added to each well. The inhibition of cell growth induced by the
tested complexes was detected by measuring the absorbance of
The increase in conductivity due to formation of acid by the each well at 515 nm (ref. 71 and 72) using a BIOTEK ELx800
released NO could also provide indication of NO generation. plate reader. The obtained data were plotted and tted using
Hence, using the head space gas of a Teon capped sealed vial GraphPad Prism 5® Ver 5.03 and shown in Fig. S5–S10.†
containing 1a in an aqueous solution with 20% DMSO the
conductivity was measured using a Mettler Toledo SevenGo
conductivity meter (Fig. S16†).
Statistical analysis
All the IC50 data are given are mean ꢁ standard deviation. The
results are mean of three independent experiments carried out
ESI-MS studies
in each cell line where, in each experiment each concentration
ESI-MS were recorded using micromass Q-Tof microTM, Waters
was assayed in triplicate. The statistical analyses were per-
and Bruker maXis Impact (ESI-QTOF) in +ve mode. The samples
were prepared in acetonitrile or methanol solution and for
mechanistic pathways determination the samples were
formed using Graph pad prism® soware 5.0 with student's
t-test.
prepared in acetonitrile–water mixture (2 : 1) and immediately
Cell cycle arrest
recorded. We even tried other aqueous mixtures viz. methanol–
water (2 : 1 v/v), DMSO–water (0.5 : 9.5 v/v). All of these solvent A549 cells (1 ꢃ 106 per plate) were grown in 100 mm dia petri
mixtures gave the same m/z pattern but the intensities were dish suspended in 12 mL DMEM media at previously
better with acetonitrile–water (2 : 1 v/v).
mentioned culturing condition. Aer 48 h, media was removed
followed by addition of fresh media. Appropriate concentration
of compound solution of 2a were added and incubated at same
condition as above. Aer 24 h drug exposure, cells were har-
vested by trypsinization, centrifuged and washed twice with
cold 1ꢃ PBS buffer (pH ¼ 7.2). Cells were again resuspended in
100 mL cold 1ꢃ PBS buffer and xed with 70% aqueous ethanol
Cell lines and culture condition
Human breast adenocarcinoma cell line (MCF-7), human lung
carcinoma cell line (A549), human embryonic kidney cell
(HEK293T) and normal mouse embryonic broblast cell line
(NIH3T3) were kindly provided by Department of Biological
Sciences, IISER-Kolkata, India. The cell lines were maintained
in the logarithmic phase at 37 ꢂC in a 5% carbon dioxide
atmosphere using a culture media containing DMEM, 10%
foetal bovine serum (GIBCO), antibiotics (100 units mLꢀ1
penicillin and 100 mg mLꢀ1 streptomycin). In hypoxic condition
the oxygen percentage was maintained at 1.5%.
ꢂ
for overnight at 4 C. DNA staining was done by resuspending
the cell pellets in 1ꢃ PBS solution containing PI (55 mg mLꢀ1
)
and RNase A (100 mg mLꢀ1) solution. Cell suspensions were
gently mixed and incubated at 37 ꢂC for half an hour. Then
samples were analyzed in a BD Biosciences FACSCalibur ow
cytometer.
Cell viability assay
Fluorescence microscopy
The cytotoxicity of compounds were evaluated on MCF-7, A549, A549 cells, 12 ꢃ 103 cells per well, were seeded in a 6-well plate
HEK293T and NIH3T3 cell lines by MTT assay.70 Briey, 6 ꢃ 103 with 3 mL of DMEM media and incubated for 48 h in afore-
cells per well, were seeded in 96-well plates in DMEM (200 mL) mentioned culture condition. Aer incubation, fresh media
and then incubated at 37 ꢂC in a 5% carbon dioxide atmo- was added and drug solutions were added. Aer 24 h of drug
sphere. Aer 48 h, the media was removed and replaced with a exposure, cells were xed with 4% paraformaldehyde solution
fresh one. Compounds to be studied were added at appropriate in 1ꢃ PBS (pH 7.2). Then the staining was performed with 300
concentrations. Each concentration was tested in triplicates in nM of DAPI solution. Aer several times washing with cold 1ꢃ
the wells. The compounds to be added were rst solubilised in PBS (pH 7.2), optical microscopy images of A549 cells were
media or PBS containing DMSO (when needed) such that the acquired using OLYMPUS IX 81 epiuorescence inverted
concentration of DMSO in well should not exceed 0.2%. BCNU microscope at 60ꢃ magnication. Both DIC and uorescence
2144 | RSC Adv., 2015, 5, 2137–2146
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microscopy images were taken and processed using OLYMPUS
Cell P soware.
7 S. K. Carter, F. M. Schabel Jr, L. Broder and T. P. Johnston,
Adv. Cancer Res., 1972, 16, 273–332.
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The N-(2-chloroethyl)-N-nitrosoureas derived from aromatic
amines or nimustine hydrochloride may involve release of NO
in their mechanistic pathway of action for anticancer activity.
The results suggest that both the compound 1a and 2a are most
active against A549 (IC50 ¼ 38 ꢁ 1 mM and 41 ꢁ 5 mM) and are
equally good even in hypoxia which is not always so
common.73,74 The aqueous decomposition rate does not neces-
sarily correlate with the in vitro cytotoxicity data. 2a arrests the
cell cycle of A549 in G2/M-phase. Nimustine which was found to
be least toxic showed a higher NO release rate even at physio-
logical pH as per the overhead gas studies. Hence, it appears
that if NO release occurs faster then the compound may become
less active but if the NO release is slow the compounds may be
more active. In addition the results suggest that the mechanistic
pathways of action of N-(2-chloroethyl)-N-nitrosoureas involves
multiple dissociative pathways and NO release is not the sole
pathway of action as suggested by earlier work in this area.
Nevertheless our work shows that NO release should be taken
into account due to its signicant occurrence at physiological
pH. The above work not only highlights a new possible mech-
anistic pathway of action for N-(2-chloroethyl)-N-nitrosoureas
but also shows that aromatic N-(2-chloroethyl)-N-nitrosoureas
may be potent anticancer agents based on their in vitro studies
in comparison to BCNU and nimustine.
12 M. Santoni, S. Scoccianti, I. Lolli, M. G. Fabrini, G. Silvano,
B. Detti, F. Perrone, G. Savio, R. Iacovelli, L. Burattini,
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Y. Sawamura, H. Kobayashi, R. Nishikawa, K. Mishima,
Y. Muragaki, T. Maruyama, J. Kuratsu, H. Nakamura,
M. Kochi, Y. Minamida, T. Yamaki, T. Kumabe,
T. Tominaga, T. Kayama, K. Sakurada, M. Nagane,
K. Kobayashi, H. Nakamura, T. Ito, T. Yazaki, H. Sasaki,
K. Tanaka, H. Takahashi, A. Asai, T. Todo, T. Wakabayashi,
J. Takahashi, S. Takano, T. Fujimaki, M. Sumi, Y. Miyakita,
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Acknowledgements
15 T. Moritz, W. Mackay, B. J. Glassner, D. A. Williams and
L. Samson, Cancer Res., 1995, 55, 2608–2614.
We sincerely acknowledge DST for the funding (Vide Project no.
SR/S1/IC-36/2010). We thank Dr Jayasri Das Sarma, Dr Tapas K
Sengupta and Dr Partho Sarothi Ray, Department of Biological
Sciences, IISER Kolkata, India for kind donation of the cell
lines. We are also thankful to IISER Kolkata for the nancial
and infrastructural support including NMR, FACS and ESI-MS,
single crystal X-ray diffraction facility. AS sincerely acknowl-
edges IISER Kolkata for research fellowship. SK and KP thank
UGC and SB thanks CSIR respectively for research fellowship.
16 C. C. Bailey, H. B. Marsden and P. H. Jones, Fatal pulmonary
brosis following 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)
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