Design, synthesis and evaluation of naphthalene-2-carboxamides as reversal agents in MDR cancer

Article abstract of DOI:10.1016/j.bmc.2006.05.010

A novel class of molecules with structure N-[3-(4-substituted-1-piperazinyl) propyl]-6-methoxy naphthalene-2-carboxamides were designed by generating a pharmacophore for potent MDR reversal activity, using Elacridar (GF 120918) as a query molecule and using MOE software. They were synthesized by condensing 6-methoxynaphthalene-2-carboxylic acid with N-[3-(4-substituted-1-piperazinyl) propyl] amines in the presence of DCC in DMF. They were evaluated in P388 murine lymphocytic leukemia cell line (P388) in vitro using SRB assay for cytotoxicity and in adriamycin-resistant P388 murine lymphocytic leukemia cell line (P388/ADR) using MTT assay for resistant reversal activity. Test compounds were non-toxic at the doses studied (upto 80 μg/ml). They effectively reversed adriamycin resistance at the doses studied (40 and 80 μg/ml). The percentage enhancement in adriamycin activity was in the range 33.58 -90.67 (at 40 μg/ml) and 8.80-46.04 (at 80 μg/ml) and the corresponding reversal potency values were in the range 1.33-1.90 and 1.08-1.46, respectively. Test compounds 2, 3, and 5 exhibited better activity as compared to the standard resistant reversal agent (Verapamil), at same concentration.

Full text of DOI:10.1016/j.bmc.2006.05.010

Bioorganic & Medicinal Chemistry 14 (2006) 6022–6026
Design, synthesis and evaluation of naphthalene-2-carboxamides
as reversal agents in MDR cancer
q
a,
a
a
b
*
Tushar N. Lokhande, C. L. Viswanathan, Advait Joshi and Aarti Juvekar
a
Department of Pharmaceutical Chemistry, Bombay College of Pharmacy, Kalina, Santacruz (E), Mumbai 400 098, India
b
Department of Chemotherapy, Tata Memorial Centre, Advanced Center for Treatment,
Research and Education in Cancer (ACTREC), Kharghar, New Mumbai 410 208, India
Received 17 March 2006; revised 4 May 2006; accepted 5 May 2006
Available online 30 May 2006
Abstract—A novel class of molecules with structure N-[3-(4-substituted-1-piperazinyl) propyl]-6-methoxy naphthalene-2-carboxa-
mides were designed by generating a pharmacophore for potent MDR reversal activity, using Elacridar (GF 120918) as a query
molecule and using MOE software. They were synthesized by condensing 6-methoxynaphthalene-2-carboxylic acid with N-[3-(4-
substituted-1-piperazinyl) propyl] amines in the presence of DCC in DMF. They were evaluated in P388 murine lymphocytic leu-
kemia cell line (P388) in vitro using SRB assay for cytotoxicity and in adriamycin-resistant P388 murine lymphocytic leukemia cell
line (P388/ADR) using MTT assay for resistant reversal activity. Test compounds were non-toxic at the doses studied (upto 80 lg/
ml). They effectively reversed adriamycin resistance at the doses studied (40 and 80 lg/ml). The percentage enhancement in adria-
mycin activity was in the range 33.58 ꢀ90.67 (at 40 lg/ml) and 8.80–46.04 (at 80 lg/ml) and the corresponding reversal potency val-
ues were in the range 1.33–1.90 and 1.08–1.46, respectively. Test compounds 2, 3, and 5 exhibited better activity as compared to the
standard resistant reversal agent (Verapamil), at same concentration.
ꢀ
2006 Elsevier Ltd. All rights reserved.
1
. Introduction
P-gp inhibitory activity and then design a novel series
of compounds, synthesize them, and evaluate them in
adriamycin-resistant P388 murine lymphocytic leukemia
cell line (P388/ADR) for resistance reversal activity.
Multidrug resistance is a serious problem in the effective
management of cancer. P-glycoprotein (P-gp), a mem-
brane protein, may be involved in the efflux of cytotoxic
1
agents from cells leading to development of resistance.
Drugs like Verapamil, Propafenone, and many others
were evaluated as reversal agents and showed promise.
However, they are non-specific in their action and pro-
2
. Results and discussion
A pharmacophore for MDR reversal activity was gener-
ated using GF120918 as a query molecule and based on
this a novel class of compounds were designed with gen-
eral structure N-substituted-6-methoxynaphthalene-2-
carboxamides (Fig. 1).
2
–4
duced toxicity when evaluated in vivo.
the development of elacridar (GF120918), which is more
This led to
5
specific in its action and is free from toxic potential.
Additionally structural features and physico-chemical
properties required for exhibiting potent P-gp inhibitory
6
activity have been identified. In the present work, it is
proposed to first generate a pharmacophore for potent
They were synthesized by an efficient synthetic process
(yield 66.8–75.7%). They were evaluated for cytotoxicity
in vitro in P388 murine lymphocytic leukemia cell line
using SRB assay and test compounds were found to
be non-toxic upto a concentration of 80 lg/ml as per-
centage growth inhibition was very low (0–10.2%).
Keywords: 6-Methoxy naphthalene-2-carboxamides; MDR reversal
agents; P-glycoprotein; Chemosensitizers.
q
This work was presented in the poster session of 4th Drug Discovery
Symposium, organized by Johnson & Johnson Pharmaceutical
Research & Development, held at Aula Major of the University of
Antwerp, Belgium, 7–8, April 2005, p 72.
Cytotoxicity evaluation data are given in Table 1.
*
0
Corresponding author. Tel.: +91 022 26670871/+91 9869163700; fax:
91 022 26670816; e-mail addresses: tusharlokhande@hotmail.com;
chelakara_viswanathan@yahoo.com
+
The % of cells killed, that is cytotoxicity index (CI), was
calculated as follows:
968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.05.010

T. N. Lokhande et al. / Bioorg. Med. Chem. 14 (2006) 6022–6026
6023
age inhibition due to test compounds+adriamycin and
that due to adriamycin alone, expressing it as percent-
age, while the reversal potency is the ratio of the per-
centage inhibition due to test compounds+adriamycin
and that due to adriamycin.
CONH
N
N
R
1
R
Compds
R
R1
The percentage enhancement in adriamycin activity was
in the range 33.58–90.67 and reversal potency was in the
range of 1.33–1.90 at 40 lg/ml dose of test compounds,
the corresponding values were 8.80–46.04% and 1.08–
1
2
3
4
5
6
-OCH3
-OCH3
-OCH3
-CH3
-n-C H9
4
1.46 at 80 lg/ml dose of test compounds.
-t-C H9
4
-OCH3
6
-C H5
Test compounds 2, 3, and 5 exhibited better activity as
compared to the standard resistant reversal agent
-OCH3
-H
(Verapamil), at same concentration.
-H
-CH3
Test compounds 1–6 were more effective as chemosensi-
tizers at the lower dose (40 lg/ml), compared to the
activity at 80 lg/ml, a quality that is desired in a good
chemosensitizing agent.
Figure 1. General structure for test compounds 1–6.
absorbance of drug treated cells
CI ¼
ꢁ 100
absorbance of control cells
3
. Conclusion
Test compounds 1–6 exhibited chemosensitizing activity
at the doses studied, and they effectively reversed adria-
mycin resistance when evaluated in vitro in adriamycin-
resistant P388 murine lymphocytic leukemia cell line
The present work led to the development of novel and
potent resistant reversal agents for MDR cancer and
three of the novel agents have shown better activity as
compared to the standard resistant reversal agent
(
P388/ADR) at 40 and 80 lg/ml (Table 2).
(Verapamil), at the same concentration.
The percentage enhancement in adriamycin activity was
calculated by considering the difference in the percent-
4. Experimental
Table 1. In vitro cytotoxicity on P388 murine lymphocytic leukemia
cell line for test compounds 1–6
4.1. Pharmacophore generation
Compound
Concentration (lg/ml)
% cell killed
Chemical Computing Group Inc.’s Molecular Operating
Environment (MOE) 2001.02 and 2002.03 versions were
used for pharmacophore generation. The computations
were done on a Windows platform with a processor of
1.6 GHz and memory 512 RAM. A database of 2000
compounds was prepared by using structures reported
in the literature and elacridar was used as a query mol-
1
10
0.0
0.0
2
4
8
0
0
0
2.3
10.2
2
3
4
5
6
10
0.0
0.0
0.0
0.0
2
4
8
0
0
0
5
ecule for pharmacophore generation. A similarity
searching protocol was applied by using elacridar as
the query molecule. This was done on the basis of the
fingerprints matching of the query molecule with each
of the compounds in the database. On the basis of
Tanimoto coefficient, best matched compounds were
short-listed. Each of these molecules was aligned with
elacridar by using MOE-FlexAlign, by taking the
descriptors in various permutations and combinations.
From the scores obtained, the important factors identi-
fied for P-gp inhibition were logP, H-bond donor,
H-bond acceptor, aromaticity, and molar volume. By
using the pharmacophoric tools, a pharmacophore was
built by using Pharmacophore Query Editor based on
the information on these short-listed molecules. The dis-
tances between the common centers (pharmacophoric
points) were measured and are given in Figure 2.
10
0.0
0.0
0.0
0.0
2
4
8
0
0
0
10
9.4
9.8
2
4
8
0
0
0
10.2
8.1
10
0.0
2.6
4.0
4.6
2
4
8
0
0
0
10
0.0
0.0
0.0
0.0
2
4
8
0
0
0
On the basis of pharmacophoric distances, a novel
naphthalene carboxamide molecule was designed. It
consists of a naphthalene ring connected by an alkyl

6024
T. N. Lokhande et al. / Bioorg. Med. Chem. 14 (2006) 6022–6026
Table 2. In vitro MDR reversal activity in P388 murine lymphocytic leukemia adriamycin-resistant cell line for test compounds 1–6
Test system
Concn lg/ml % cell killed % enhancement Reversal Concn lg/ml % cell killed % enhancement in Reversal
(lmol)
in ADR activity potency (lmol)
ADR activity
potency
Compd-1
ADR
Compd-1 + ADR 80 + 60
80 (0.234)
60
1.80
24.43
35.68
40 (0.117)
50
40 + 50
2.30
17.48
27.13
46.04
44.31
39.58
32.62
31.60
8.80
1.46
1.40
1.39
1.32
1.31
1.08
1.90
55.20
1.55
Compd-2 80 (0.208)
ADR 60
Compd-2 + ADR 80 + 60
1.30
24.43
34.28
40 (0.104)
50
40 + 50
2.23
17.48
32.85
87.92
1.87
Compd-3 80 (0.208)
ADR 60
Compd-3 + ADR 80 + 60
3.15
24.43
34.10
40 (0.104)
50
40 + 50
3.33
17.48
33.33
90.67
1.90
Compd-4 80 (0.198)
ADR 60
Compd-4 + ADR 80 + 60
2.10
24.43
32.40
40 (0.099)
50
40 + 50
2.05
17.48
29.78
70.36
1.70
Compd-5 80 (0.244)
ADR 60
Compd-5 + ADR 80 + 60
1.80
24.43
32.15
40 (0.122)
50
40 + 50
1.63
17.48
30.83
76.37
1.76
Compd-6 80 (0.256)
ADR 60
Compd-6 + ADR 80 + 60
0.00
24.43
26.58
40 (0.128)
50
40 + 50
0.30
17.48
23.35
33.58
1.33
VRP
ADR
VRP + ADR
80 (0.162)
60
80 + 60
10.8
27.6
52.5
40 (0.081)
50
40 + 50
5.1
27.6
47.3
90.21
71.37
1.71
ADR, Adriamycin; VRP, Verapamil.
1
linker to N of a piperazine ring. The alkyl linker was
optimized to a propyl group to satisfy the pharmaco-
phoric distances. Substitutions were then made (main-
mide with mono-substituted piperazines (N-methyl
piperazine, N-butyl piperazine, N-tert-butyl piperazine,
and N-phenyl piperazine) and unsubstituted piperazine
in DMF in the presence of anhydrous K CO , followed
4
taining the pharmacophoric distances) on the N of
piperazine to achieve the desired lipophilicity for P-gp
inhibition.
2
3
by reaction with hydrazine hydrate and acidification
with HCl. After removal of phthalyl hydrazide, the
filtrate was made alkaline and the free amine was
extracted in chloroform/ethyl acetate mixture (1:2).
4
.2. Chemistry
8
Removal of solvent gave the desired amines.
All melting points were recorded on Thermonik melting
point apparatus and are uncorrected; IR spectra were
recorded in Jasco FT/IR 5300 spectrophotometer; H
2-Naphthoic acid required for the synthesis of com-
pound 6 was prepared from naphthalene by acetylation
1
NMR spectra were recorded in Varian spectrophotome-
ter at 300 MHz in CDCl (TMS as internal reference).
Column chromatography was performed using Silica
gel, 60–120 mesh, Qualigens fine chemicals, India.
Elemental analyses values for final compounds were
within ±0.4% of theoretical value.
using acetyl chloride and anhydrous AlCl in nitroben-
3
9
zene, followed by oxidation using sodium hypobromite
9,10
3
10
(NaOBr). Yield: 60%; mp: 182–184 ꢁC (184–185 ꢁC).
IR (KBr, cm ): 3053, 2839 (C–H stretch), 2646, 2569 (O–
ꢀ
1
H stretch), 1686 (C@O stretch).
2
-Naphthoic acid was condensed with N-[3-(4-methyl-
N-[3-(4-Substituted-piperazinyl) propyl]-6-methoxy
naphthalene-2-carboxamides were prepared by condens-
ing 6-methoxy-2-naphthoic acid with N-[3-(4-substitut-
ed-1-piperazinyl) propyl] amines in the presence of
DCC in N,N-dimethylformamide (DMF) as solvent.
1-piperazinyl) propyl] amine in the presence of DCC
in DMF to obtain N-[3-(4-methyl-1-piperazinyl) propyl]
naphthalene-2-carboxamide (compound 6) (Section
4.2.8).
6
6
-Methoxy-2-naphthoic acid was prepared by oxidizing
-methoxy-2-naphthaldehyde with ammonical silver
4.2.1. Synthesis of 6-methoxy-2-naphthoic acid. To 20 g
(115 mM) of silver nitrate in 400 ml of distilled water,
7.5 ml of 10% sodium hydroxide was added, followed
by addition of 2% ammonia solution to dissolve the
silver oxide. To the solution 20 g (105 mM) of 6-meth-
oxy-2-naphthaldehyde was added and stirred for 5 h with
temperature maintained at 50–60 ꢁC. The solution was
concentrated, cooled to room temperature and the prod-
uct formed was digested with 3· 75 ml of 10% sodium
hydroxide solution. Extract on acidification gave the free
acid, which was purified by recrystallization from alcohol.
nitrate. 6-Methoxy-2-naphthaldehyde was prepared
from 2-naphthol by bromination followed by methyla-
tion using dimethyl sulfate in alkaline condition. The
dry product was then treated with Mg in dry THF to
form a Grignard reagent, which on reaction with
7
DMF gave the aldehyde.
The N-[3-(4-substituted-1-piperazinyl) propyl] amines
were prepared by reacting N-(3-bromopropyl) phthali-

T. N. Lokhande et al. / Bioorg. Med. Chem. 14 (2006) 6022–6026
6025
Figure 2. Pharmacophore (Acc, acceptor; Don, donor; Aro, aromatic).
1
1
Yield: 60%; mp: 203–204 ꢁC (201–202ꢁC). IR (KBr,
cm ): 3013, 2966, 2924 (C–H stretch), 2845, 2565 (O–H
(1H, s, –NH), 7.1–7.2 (2H, m, Ar-H), 7.6 (1H, s, Ar-
H), 7.8 (2H, dd, Ar-H), 8.0 (1H, s, Ar-H).
ꢀ
1
stretch), 1686 (C@O stretch), 1209 (C–O stretch, ether)
1
H NMR (CDCl ) d ppm: 3.96 (3 H, s, –OCH ), 7.17–
4.2.5. N-[3-(tert-Butyl-1-piperazinyl) propyl]-6-methoxy
naphthalene-2-carboxamide (3). Yield: 67.4%; mp 135–
3
3
7.26 (3· 1H, m, Ar-H), 7.78 (1H, d, Ar-H), 7.89 (1H, d,
Ar-H), 8.10 (1H, dd, Ar-H), 8.63 (1H, s, –COOH).
ꢀ1
137 ꢁC. IR (KBr, cm ): 3296 (N–H stretch), 1707
1
(
–
C@O stretch); H NMR (CDCl ) d ppm: 1.2 (9H, s,
3
C(CH ) ), 1.5 (2H, m, –(CH )–, propyl), 1.8 (8H, m,
3 3 2
4
.2.2. General method for preparation of N-[3-(4-
substituted-1-piperazinyl) propyl]-6-methoxy naphtha-
lene-2-carboxamides. 6-Methoxy-2-naphthoic acid
10 mM) was condensed with five N-[3-(4-substituted-
piperazine), 2.8 (4H, m, 2· –CH –, propyl), 3.8 (3H, s,
2
–
OCH ), 6.2 (1H, s, –NH), 7.1–7.2 (2H, m, Ar-H), 7.6
3
(1H, s, Ar-H), 7.8 (2H, dd, Ar-H), 8.0 (1H, s, Ar-H).
(
1
-piperazinyl) propyl] amines (12 mM) in turn in the
4.2.6. N-[3-(4-Phenyl-1-piperazinyl) propyl]-6-methoxy
naphthalene-2-carboxamide (4). Yield: 66.8%; mp 138–
presence of DCC (12 mM) in DMF to get compounds
–5. Dicyclohexylurea formed was removed by filtra-
1
ꢀ1
1
40 ꢁC. IR (KBr, cm ): 3287 (N–H stretch), 1707
tion and DMF was removed under vacuum. The res-
idue obtained was washed with water to remove
excess of amine and traces of DMF. The residue
was purified by column chromatography using chloro-
form/ethyl acetate, 80:20 as eluent and then recrystal-
lized from alcohol.
1
(C@O stretch); H NMR (CDCl ) d ppm: 1.5 (2H, m,
3
–
2
–
(CH )–, propyl), 1.8 (8H, m, piperazine), 2.8 (4H, m,
· –CH –, propyl), 3.8 (3H, s, –OCH ), 6.2 (1H, s,
2
2
3
NH), 6.5 (2H, d, Ar-Ph), 6.60 (1H, t, Ar-Ph), 6.80
(2H, t, Ar-Ph), 7.1–7.2 (2H, m, Ar-H),7.6 (1H, s, Ar-
H), 7.8 (2H, dd, Ar-H), 8.0 (1H, s, Ar-H).
4.2.3. N-[3-(4-Methyl-1-piperazinyl) propyl]-6-methoxy
naphthalene-2-carboxamide (1). Yield: 70.2%; mp: 137–
4
.2.7. N-[3-(1-Piperazinyl) propyl]-6-methoxy naphtha-
lene-2-carboxamide (5). Yield: 75.7%; mp 143–145 ꢁC.
ꢀ
1
1
38 ꢁC. IR (KBr, cm ): 3327 (N–H stretch), 1684
ꢀ1
IR (KBr, cm ): 3288 (N–H stretch), 1707 (C@O
1
(
C@O stretch); H NMR (CDCl ) d ppm: 1.5 (2H, m,
1
3
stretch); H NMR (CDCl ) d ppm: 1.5 (2H, m,
3
–
–
–
(CH )–, propyl), 1.8 (8H, m, piperazine), 2.2 (3H, s,
CH ), 2.8 (4H, m, 2· –(CH )–, propyl), 3.8 (3H, s,
2
–
–
3
(CH )–, propyl), 1.8 (8H, m, piperazine), 2.1 (1H, s,
N–H–, piperazine), 2.8 (4H, m, 2· –CH –, propyl),
2
3
2
2
OCH ), 6.2 (1H, s, –NH), 7.1–7.2 (2H, m, Ar-H), 7.6
3
.9 (3H, s, –OCH ), 6.2 (1H, s, –NH, amide), 7.1–7.2
3
(
1H, s, Ar-H), 7.8 (2H, dd, Ar-H), 8.0 (1H, s, Ar-H).
(2H, m, Ar–H), 7.6 (1H, s, Ar-H), 7.8 (2H, dd, Ar-H),
8.0 (1H, s, Ar-H).
4.2.4. N-[3-(4-Butyl-1-piperazinyl) propyl]-6-methoxy
naphthalene-2-carboxamide (2). Yield: 68%; mp 130–
4.2.8. N-[3-(4-Methyl-1-piperazinyl) propyl]-naphthalene-
2-carboxamide (6). Yield: 71.2%; mp 184–185 ꢁC. IR
ꢀ
1
1
32 ꢁC. IR (KBr, cm ): 3283 (N–H stretch), 1707
1
ꢀ1
(
C@O stretch); H NMR (CDCl ) d ppm: 0.8 (3H, t,
(KBr, cm ): 3327 (N–H stretch), 1684 (C@O stretch);
3
1
CH , n-butyl), 1.2 (6H, m, 3· –(CH )–, n-butyl), 1.5
H NMR (CDCl ) d ppm: 1.5 (2H, m, –(CH )–, propyl),
3
2
3
2
(2H, m, –(CH )–, propyl), 1.8 (8H, m, piperazine), 2.8
(4H, m, 2· –(CH )–, propyl), 3.8 (3H, s, –OCH ), 6.2
1.8 (8H, m, piperazine), 2.2 (3H, s, –CH ), 2.8 (4H, m,
2· –CH –, propyl), 6.2 (1H, s, –NH), 7.10–7.29 (2H,
2
3
2
3
2

6026
T. N. Lokhande et al. / Bioorg. Med. Chem. 14 (2006) 6022–6026
m, Ar-H), 7.67 (2H, d, Ar-H), 7.8 (2H, dd, Ar-H), 8.0
1H, s, Ar-H).
fixed with 50 ll of cold (4 ꢁC) 50% trichloroacetic acid
(TCA) and were further incubated at 37 ꢁC for 1 h,
washed five times with distilled water, and plates were
air-dried and stored until use. The TCA fixed cells were
stained for 30 min with SRB reagent, at the end excess
stain was removed by quickly rinsing the cells four times
with 1% acetic acid. Residual wash solution was re-
moved by sharply flicking the plates over the sink. The
wells were air-dried until no moisture is visible. The
bound dye was solubilized in 100 ll of 10 mM unbuf-
fered Tris base and absorbance of the solution was
recorded on spectrophotometer at a wavelength of
565 nm.
(
4
.3. Pharmacological evaluation
Test compounds were evaluated for cytotoxicity in vitro
in P388 murine lymphocytic leukemia cell line (P388)
1
2
using SRB assay and resistance reversal activity in vitro
in adriamycin-resistant P388 murine lymphocytic leuke-
1
3
mia cell line (P388/ADR) using MTT assay.
Parental P388 cell line was grown and maintained in the
BDF mice by intraperitoneal weekly transplantation of
1
the cells. Resistant cell line (P388/ADR) was developed
by giving intraperitoneal injections of 0.65 mg/kg adria-
mycin with days 1–9 schedule, for 20 passages in BDF1
mice. For evaluation, tumor cells were obtained from
mice bearing seven-day-old tumor. The ascitic fluid from
the intraperitoneal cavity of donor mice was aspirated
asceptically into 0.9% sodium chloride. The final cell
Acknowledgments
Authors are thankful to All India Council for Technical
Education (AICTE), New Delhi, and Indian Pharma-
ceutical Association—Shri. Ramanbhai B. Patel founda-
tion (IRF), Mumbai, for financial support.
7
density was adjusted to 10 cells/ml.
4
.3.1. Preparation of cell suspension. Culture cells were
suspended in sterile saline and centrifuged at 1000 rpm
for 10 min. The tumor cells were subjected to cold hypo-
tonic shock (4 ꢁC) of ammonium chloride buffer (pH
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(
5
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centration 5 · 10 cells/ml.
Test compounds 1–6 were evaluated for cytotoxicity at
1
4
5
0, 20, 40 and 80 lg/ml. MDR reversal activity was
evaluated at 40 and 80 lg/ml, and the corresponding
adriamycin concentrations were 50 and 60 lg/ml,
respectively. Verapamil (VRP) was used as a standard
chemomodulator and was evaluated at 40 and 80 lg/
ml. All experiments were carried out in quadruplicate.
7
8
9
. Robarge, M. J.; Husbands, S. M.; Kieltyka, A.; Brodbeck,
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.3.2. MTT assay. Cell suspension 90 ll/well was added
to 96 wells and incubated at 37 ꢁC for 24 h in 5% CO
2
3
175.
incubator. Ten microlitres of either solutions of test
compounds or solution of adriamycin or combination
of a solution of adriamycin and solution of test com-
pounds was added to the wells and the plates were incu-
. Furniss, B. S., Hannaford, A. J., Smith, P. W. G.,
Tatchell A. R. Eds.; Vogel’s Textbook of Practical
Organic Chemistry; 5th ed.; Longman: UK, 1989; pp
1010, 1064–1065.
bated at 37 ꢁC for 48 h in 5% CO incubator. MTT
10. Newman, M. S.; Holmes, H. L.. In Organic Synthesis;
Blatt, N. Y., Ed.; John Wiley and Sons: New York, 1943;
Vol. 2, pp 428–430.
2
reagent 10 ll was then added to the wells and further
incubated for 6 h. The supernatant was then carefully
removed and the formazan crystals were dissolved in
1
1. Itoh, k.; Miyake, A.; Tanabe, M.; Hirata, M.; Oka, Y.
Chem. Pharm. Bull. 1983, 3, 2006.
100 ll of acidified isopropanol (0.04 N HCl in isopropa-
nol) and the absorbance of the solution was then read on
spectrophotometer at a wavelength of 540 nm.
1
2. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.;
McMohan, J.; Vistica, D.; Warren, J. T.; Bokesch, H.;
Kenney, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82,
1
107.
4.3.3. SRB assay. Procedure was same upto addition of
test solutions to the microtiter plate. The cells were then
13. Twentyman, P. R.; Fox, N. E.; Rees, J. K. H. Br. J.
Haematol. 1989, 71, 19.