Supplementary Information
Synthesis, Characterization and Cytotoxicity Evaluation of an Oleic Acid
derived Novel Bicephalous Dianionic Surfactant
Rahul S. Kalhapure† and Krishnacharya G. Akamanchi*
Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology,
Matunga, Mumbai- 400019, India.
*Correspondence: Tel.:+91-22-33612214; Fax: 91-22-33611020.
E-mail:kgap@rediffmail.com; kg.akamanchi@ictmumbai.edu.in (K.G. Akamanchi).
†
Current address: Discipline of Pharmaceutical Sciences, School of Health Sciences,
University of KwaZulu-Natal, Private Bag X54001, Durban 4000, South Africa.
S1
Analytical
data
of
intermediates
in
the
synthesis
of
disodium
(Z)
3,
3’-
(oleoylazanediyl)dipropanoate------------------------------------------------------------------------------- S3
Fig. S1. 13C NMR spectra of (Z)-dimethyl 3,3’-(oleoylazanediyl)dipropanoate 4-----------------
S3
Fig. S2. ESI-MS spectra of (Z)- 3,3’-(oleoylazanediyl)dipropanoic acid 5-------------------------- S4
Fig. S3. Graph of ml of NaOH versus potential----------------------------------------------------------
S5
Table S1. Values obtained for (Z)- 3,3’-(oleoylazanediyl)dipropanoic acid 5 from pH metry-
S5-S6
% Purity calculations of (Z)- 3,3’-(oleoylazanediyl)dipropanoic acid 5----------------------------- S6
SRB assay procedure------------------------------------------------------------------------------------------
S2
S7-S9
3,3’-(benzylazanediyl)dipropanoate 2
FT-IR (neat) ν: 2947(vs), 2822(s), 2044(m), 1954(m), 1876, 1742, 1728(s), 1601(s),
1176(m), 1036(m) cm-1. 1H NMR (60 MHz, CDCl3) δ: 2.56 (m, 4H, -CH2CO-), 2.95 (m, 4H,
-NCH2-), 3.59 (s, 2H, Ar-CH2-), 3.64 (s, 6H, -COOCH3), 7.27 (m, 5H, Ar).
Dimethyl 3,3’-azanediyldipropanoate 3
FT-IR (neat) ν: 3372(br), 2958(s), 2924(s), 1724(s), 1612(m), 1170(m), 1028(m) cm-1. 1H
NMR (60 MHz, CDCl3) δ: 2.60 (t, 4H, J = 11.76 Hz, -CH2CO-), 2.99 (t, 4H, J = 11.46 Hz, NH-CH2-), 3.68 (s, 6H, -COOCH3).
Fig. S1. 13C NMR spectra of (Z)-dimethyl 3,3’-(oleoylazanediyl)dipropanoate4
S3
Fig. S2. ESI-MS spectra of (Z)- 3,3’-(oleoylazanediyl)dipropanoic acid 5
S4
Fig. S3. Graph of ml of NaOH versus potential.
Table S1. Values obtained for (Z)- 3, 3’-(oleoylazanediyl)dipropanoic acid 5 from pH metry
(n= 3).
ml of 0.04 N NaOH added
pH
∆V
∆pH
∆pH/∆V
0
3.68 ± 0.08
0.2
0.16 ± 0.02
0.8 ± 0.1
0.2
3.84 ± 0.10
0.2
0.17 ± 0.05
0.85 ± 0.25
0.4
4.01 ± 0.05
0.2
0.19 ± 0.01
0.95 ± 0.05
0.6
4.20 ± 0.06
0.2
0.13 ± 0.03
0.65 ± 0.15
0.8
4.33 ± 0.03
0.2
0.11 ± 0.01
0.55 ± 0.05
1
4.44 ± 0.02
0.5
0.22 ± 0.07
0.44 ± 0.14
1.5
4.66 ± 0.09
0.5
0.14 ± 0.02
0.28 ± 0.04
2
4.80 ± 0.07
0.5
0.14 ± 0.01
0.28 ± 0.02
2.5
4.94 ± 0.06
0.5
0.11 ± 0.03
0.22 ± 0.06
3
5.05 ± 0.09
0.5
0.08 ± 0.06
0.16 ± 0.12
3.5
5.13 ± 0.03
0.5
0.08 ± 0.01
0.16 ± 0.02
4
5.21 ± 0.02
0.5
0.09 ± 0.04
0.18 ± 0.08
4.5
5.30 ± 0.06
0.5
0.07 ± 0.01
0.14 ± 0.02
5
5.37 ± 0.05
0.5
0.06 ± 0.05
0.12 ± 0.1
5.5
5.43 ± 00
0.5
0.07 ± 0.05
0.14 ± 0.1
S5
6
5.50 ± 0.05
0.5
0.07 ± 0.01
0.14 ± 0.02
6.5
5.57 ± 0.04
0.5
0.06 ± 0.05
0.12 ± 0.1
7
5.63 ± 0.09
0.5
0.07 ± 0.06
0.14 ± 0.12
7.5
5.70 ± 0.03
0.5
0.07 ± 0.01
0.14 ± 0.02
8
5.77 ± 0.02
0.5
0.10 ± 0.03
0.2 ± 0.06
8.5
5.87 ± 0.05
0.5
0.13 ± 0.04
0.26 ± 0.08
9
6.00 ± 0.01
0.5
0.16 ± 0.01
0.32 ± 0.02
9.5
6.16 ± 0.02
0.5
0.25 ± 0.02
0.50 ± 0.04
10
6.41 ± 0.04
0.5
0.29 ± 0.01
0.58 ± 0.02
10.5
6.70 ± 0.03
0.5
0.26 ± 000
0.52 ± 000
11
6.96 ± 0.03
0.5
0.35 ± 0.01
0.70 ± 0.02
11.5
7.31 ± 0.02
0.5
0.34 ± 0.02
0.68 ± 0.04
12
7.65 ± 0.04
0.5
0.63 ± 0.01
1.26 ± 0.02
12.5
8.28 ± 0.03
0.5
0.61 ± 0.02
1.22 ± 0.04
13
8.89 ± 0.01
0.5
0.33 ± 000
0.66 ± 000
13.5
9.22 ±0.01
0.5
0.18 ± 000
0.36 ± 000
14
9.40 ± 0.01
0.5
0.14 ± 000
0.28 ± 000
14.5
9.54 ± 0.01
0.5
0.08 ± 0.02
0.16 ± 0.04
15
9.62 ± 0.03
0.5
0.09 ± 0.03
0.18 ± 0.06
15.5
9.71 ± 0.06
0.5
0.05 ± 0.03
0.10 ± 0.06
16
9.76 ± 0.09
0.5
0.07 ± 0.03
0.14 ± 0.06
16.5
9.83 ± 0.06
0.5
0.05 ± 0.01
0.10 ± 0.02
17
9.88 ± 0.07
0.5
0.05 ± 0.01
0.10 ± 0.02
17.5
9.93 ± 0.08
0.5
0.04 ± 0.08
0.08 ± 0.16
% Purity calculations of (Z)- 3,3’-(oleoylazanediyl)dipropanoic acid 5
Mol. Wt. of 5 = 425.60
Therefore factor is:
Each ml of 0.04 N NaOH = 0.0085 g of 5
Therefore 12 ml of 0.04 N NaOH = 12 x 0.0085 g = 0.102 g of 5
Quantity of 5 taken was 0.105 g
Therefore % purity = [0.102/0.105] × 100 = 97%.
SRB assay procedure
Medium: Roswell Park Memorial Institute medium (RPMI 1640) containing 10% foetal
bovine serum and 2 mM/L-glutamine was used to grow cell lines.
Cell lines: Human cervix cancer cell line (HeLa).
S6
Inoculation: Inoculation of cells was carried out into 96 well microtiter plates in 100 µl at
plating density of approximately 5000 cells/well.
Incubation: Three sets of microtiter plates were incubated at 37 °C, 5 % CO2, 95 % air and
100 % relative humidity for 24 h prior to the addition of test substances and out of which
i) One set was fixed in situ with trichloroacetic acid (TCA), and processed as described under
termination of assay (point No.1 - 6) to represent a measurement of the cell population at the
time of test compound addition (Tz).
ii) Second set was incubated further for 48 h (37 oC) without adding any substance and
processed as described under termination of assay (point No.1 - 6) to represent control
growth (C) = negative control
iii) Third set was used for test substances. The test substances [adriamycin or sodium (Z) 3,
3’-(oleoylazanediyl)dipropanoate)]were added and incubated for 48 h (37 oC).
Preparation of test substances: Sodium (Z) 3, 3’-(oleoylazanediyl)dipropanoate6and
adriamycin were solubilized in distilled water and dimethyl sulfoxide respectively at100
mg/ml concentration. Both the solutions were diluted to 1 mg/ml using milli-Q water and
stored frozen prior to use.
Assay:
Test substances addition: At the time of addition, an aliquot of frozen concentrate (1mg/ml)
was thawed and diluted to 100 μg/ml, 200 μg/ml, 400 μg/ml and 800 μg/ml with complete
medium containing test article. The required final concentrations (10 μg/ml, 20 μg/ml, 40
μg/ml, 80 μg/ml) were obtained by adding aliquots of 10 µl of different dilutions to the
appropriate microtiter wells already containing 90 µl of medium.
Termination of assay: Assay was terminated by the addition of cold TCA after the
incubation of plates for 48 h at standard conditions.
1. Cells were fixed in situ by the gentle addition of 50 µl of cold 30 % (w/v) TCA (final
concentration, 10 % TCA) and incubated for 60 min at 4 °C.
2. The plates were washed five times with milli-Q water and air dried after discarding
the supernatant.
3. SRB solution (50 µl) at 0.4 % (w/v) in 1 % acetic acid was added to each of the
wells, and plates were incubated for 20 min at room temperature.
4. Unbound dye was recovered and1 % acetic acid was used to remove the residual dye.
5. The plates were air dried and bound stain was subsequently eluted with 10 mM
trizma base.
S7
6. The absorbance was read on a plate reader at a wavelength of 540 nm with 690 nm
reference wavelength.
Calculations: Percentage growth was calculated on a plate-by-plate basis for test wells
relative to control wells (n =3; the experiments were repeated thrice with 6 replicates each
time) and expressed as the ratio of average absorbance of the test wells to the average
absorbance
of
the
control
wells
x
100.
Table S2. % Growth values obtained from SRB assay (n =3).
Sample
sodium (Z) 3, 3’-(oleoylazanediyl)dipropanoate)
Adriamycin
Concentrati
10
20
40
80
10
20
40
80
99.7 ± 0.46
95.3 ± 5.80
96.7 ± 4.31
98.9 ± 1.79
-13.0±13.51
-27.8±6.43
-34.1±12.2
-36.5 ± 1.79
on (µg/ml)
% Growth
1. The percentage growth was calculated at each of the concentration levels using the six
absorbance measurements [time zero (Tz), control growth (C), and test growth in the
presence of test compounds at the four concentration levels (Ti)].
Percentage growth inhibition was calculated as:
(Ti−Tz)
% Growth = (C−Tz) 𝑋 100 for concentrations for which Ti>/=Tz. (Ti-Tz) positive or
zero.
% Growth =
(Ti−Tz)
(Tz)
𝑋 100 for concentrations for which Ti<Tz. (Ti-Tz) negative.
2. The dose response parameters were calculated for each test article. Growth inhibition
of 50 % (GI50) [concentration resulting in a 50 % reduction in the net protein increase
(as measured by SRB staining)was calculated from:
(Ti − Tz)
𝑋 100 = 50
(C − Tz)
3. The concentration resulting in total growth inhibition (TGI) was calculated from Ti =
Tz.
4. The LC50 [concentration of drug resulting in a 50% reduction in the measured protein
at the end of the drug treatment (concentration of drug causing lethality to 50 % of the
cells) as compared to that at the beginning] indicating a net loss of cells following
treatment is calculated from following equation:
S8
(Ti − Tz)
𝑋 100 = −50
(Tz)
S9