Phenacyl group containing amide derivative of dehydroabietylamine exhibiting enhanced cytotoxic activity against PLC and MCF7 cancer cell lines

Article abstract of DOI:10.1007/s00044-017-1859-0

Abstract: New amide derivatives of (+)-dehydroabietylamine, tricyclic abietane diterpene amine, were prepared using Zhongping’s protocol. (+)-N-(2-phenyl-acetyl)-dehydroabietylamine derivative (11) demonstrated noticeable growth attenuation of hepatocellu

Full text of DOI:10.1007/s00044-017-1859-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2017) 26:1367–1376
DOI 10.1007/s00044-017-1859-0
ORIGINAL RESEARCH
Phenacyl group containing amide derivative of
dehydroabietylamine exhibiting enhanced cytotoxic activity
against PLC and MCF7 cancer cell lines
Muhammad Ayaz Mustufa1,4 Afshan Aslam2 Cigdem Ozen3 Imran Ali Hashmi2
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Naim ul Hasan Naqvi1 Mehmet Ozturk3 Firdous Imran Ali2
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Received: 27 April 2015 / Accepted: 2 March 2017 / Published online: 16 March 2017
© Springer Science+Business Media New York 2017
Abstract New
amide
derivatives
of
(+)-dehy-
Graphical Abstract
droabietylamine, tricyclic abietane diterpene amine, were
prepared using Zhongping’s protocol. (+)-N-(2-phenyl-
acetyl)-dehydroabietylamine derivative (11) demonstrated
noticeable growth attenuation of hepatocellular carcinoma
cell lines including PLC/PRF/5, SNU475, Hep3B-TR, and
Huh7 with IC50 of 7.4 µM, 9.8 µM, 11.7 µM, and 11.8 µM,
respectively. A breast cancer cell line MCF7 was the most
sensitive against amide 11 with lowest IC50 value (4.8 µM).
Low cell confluence and increase in G2/M phase was
recorded after 48 and 72 h of treatment of amide 11 on PLC/
PRF/5 cell line. Finally, amide 11 has comparatively suf-
ficient therapeutic role due to addition of N-phenacyl group
at C-18. Amide 11 demonstrated as potential candidate for
future cancer interference and research.
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Keywords Cancer
Dehydroabietylamine
Amides
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Cytotoxic activity NMR spectroscopy FTIR
Introduction
The disease of our century is cancer (Dalitsch and Vazirani
1959). Cancer is the second largest cause of mortality.
Hepatocellular carcinoma (HCC) is one of the major con-
cern due to its life threatening and fetal effects. Since 1940
to 2007, out of 155 potentially active anticancer trial drugs
more than 110 were derived from natural plants and their
derivatives (Newman and Cragg 2007). Resin acids
including abietic acid, dehydroabietic acid, and analogous
known to exhibit a wide range of biological activities
(Lin et al. 2012), including cytotoxic activity against breast
cancer, prostate cancer, and liver cancer cells.
Considering that Structural manipulation is still sig-
nificant and principally one of the focused technique
employed to enhance medicinal properties of natural com-
pounds; a series of dehydroabietylamine (DAA, 1) derivatives
(amides 2–13) has been prepared by modifying C-18 amino
moiety of DAA (1) to furnish a new functionality, i.e., amide.
* Muhammad Ayaz Mustufa
ayazbukero@gmail.com
a_m_bukero@yahoo.com
* Firdous Imran Ali
firdous@uok.edu.pk
1
Baqai Institute of Pharmaceutical Sciences, Baqai Medical
University, Karachi 74600, Pakistan
2
Depatment of Chemistry, University of Karachi, Karachi, Pakistan
3
Izmir International Biomedicine and Genome Institute, iBG-izmir,
Dokuz Eylül University, 35340 Balcova-Izmir, Turkey
4
Pakistan Health Research Council, Islamabad, Pakistan

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Med Chem Res (2017) 26:1367–1376
The cytotoxicity of synthesized amides was tested against
four HCC cell lines and one breast cancer cell line.
Absorbance in terms of optical density (OD) for each
sample was measured by using microplate reader in
between SRB dye reference range. The SRB assay is non-
destructive, very stable, and colorimetric screening protocol
(Skehan et al. 1990; Vichai and Kirtikara 2006).
Amide group was selected for derivatization because of
reported biological significance of both amides and fatty
acid amides (Farrell and Merkler 2008; Koga et al. 1997;
Movahed et al. 2005). According to various pharmacolo-
gical (Farrell and Merkler 2008) and biochemical studies,
fatty acid amides act as biological lipids, found in cer-
amides, sphingo lipids, and some compounds of brain and
other tissues also possess this moiety.
Fatty acid amides are involved in many organ systems
such as immune energy balance, food intake, metabolic
homeostasis, cardiovascular depressive effects, fertility, pain,
and neuroprotection(Di Marzo 2008; Micale et al. 2007;
Pillarisetti et al. 2009). Fatty acid amides effect cell pro-
liferation (Burstein and Salmonsen 2008). Fatty acid amides
also showed anti-tuberculosis activity (D’Oca et al. 2010).
Amide formation is an important process frequently used
for a wide range of organic syntheses. Different methods for
the synthesis of amide has been reported (Azumaya et al.
2003; Burés et al. 2009; Hardee et al. 2010; Huang et al.
2007; Larrivée-Aboussafy et al. 2009; Li and Xu 1999;
Sheehan and Hess 1955; Terada et al. 2008). In the present
work, the amide moiety was generated using Huang
Zhongping’s protocol (Huang et al. 2007).
In current study, SRB method was used for screening.
HCC (Huh7) and breast cancer (MCF7) cell lines were used
for initial screening of trial drugs. For both Huh7 and MCF7
cell lines, 2000 cells/well were cultured in 96-well plate in
an incubator at 370C with 5% CO2 in complete medium
(Dulbecco's modified eagle medium, 10% fetal bovine
serum, 1% NEA, 1% L-glutamine and 1% P/S) and wait for
24 h. After 24 h of cell culturing, all trial drugs were
introduced in two different (50 µM and 20 µM) concentra-
tions in triplicate for each sample. Plates were further
incubated for next 72 h. After 72 h, media were discarded
and cells were washed once by using 1XPBS. For fixation,
50 μl of ice cold 10% Tcll and kept in dark at 40C for 1 h.
After fixation, the TCA was removed by tapping and plates
were washed 4–5 X with ~ 200 μl dH2O. Plates were left
over night for drying under hood. Finally, 50 μl of 0.4%
(SRB) in 1% acetic acid solution was added to each well
and plates left in dark at room temperature for 10 min. SRB
was removed and plates were washed 4–5 times with 1%
acetic acid before air-drying. Bound SRB was solubilized
with 100 µl of 10 mM un-buffered chilled tris-base solution
and plates were left on a plate shaker for at least 1–2 min.
Absorbance was recorded using μ-Quant microplate reader
with a wave length range of 405–515 nm. The test OD
values were defined as the absorbance of each sample.
Mean values were determined and standard deviation was
found satisfactory upto 0.25 in each respective triplicate
sample, calculated automatically using Excel 7.0 software
for Windows 2007.
Materials and methods
Biology
Stock solution preparation
All stock solutions were prepared in 100% dimethyl sulf-
oxide (DMSO) with a concentration of 20 mM. Further
dilutions were made up to 0.1% of DMSO with the help of
respective media used for each cell line.
Cell morphology
6-well plates were used for photographs and 3 × 105 cells
were maintained in each well. After 24 h drug was admi-
nistered. Photographs of most sensitive HCC (PLC/PRF/5)
cell line were taken after three consecetive intervels, i.e., 24,
48, and 72 h for both amide 11 treated and untreated cells
before and and after fixation. All photographs were taken at
20X using light microscope.
Cell lines
Ten HCC cell lines including Huh7, Hep3B, Hep3B-TR,
SNU449, Mahlavu, PLC/PRF/5, SNU475, Hep40,
SNU387, and SNU475. While, one breast cancer line
MCF7 was also used for primary screening.
Cytotoxicity screening protocol
Cell prolifration assay
Sulforhdamine B (SRB) is an amino xanthene dye with two
phenolic groups, responsible to attach with the cell proteins
in slightly acidic environment, therefore 10% trichloroacetic
acid (TCA) is used to fix the attached with protein content
of cells by maintaining acidic condition; it become solubi-
lized in basic phase by using 10 mM trisma base.
Cells were plated in 10 cm2 Petri plates at 3–4 × 105 per
plate. After drug treatment, cells were harvested in different
intervels by trypsinization and washed with PBS. Cells were
fixed in ice-cold 70% ethanol, washed, and resuspended in
3 ml of 70% ethanol for storage at 4 oC;fixed cells treated
with RNase A ; and stained with propidium iodide for 45

Med Chem Res (2017) 26:1367–1376
1369
min at room temperature. The stained cells were analyzed
(+)-N-(tetradecanoyl)-dehydroabietylamine (3)
by flow cytometry using BD FACScalibur.
Rf: 0.607(hexane/ethylacetate, 9:1) State: gummy, Yield:
89.74%, [α]D26: −15.23 (C=0.0504, CHCl3), HRMS-ESI:
calcd for C34H58NO (M+H)+ 496.4518, found 496.4281,
IR (cm-1): 3303 (N–H stretching), 2924 (C–H), 2854
(C–H), 1646 (C=O), 1551 (N–H bending), 1459 (CH2
bending), 1379 (CH3 bending), 719 (CH2 long chain band).
Chemistry
All reagents were purchased from Sigma–Aldrich and
Merck and were used without further purification. Technical
grade solvents were used for chromatography and distilled
prior to use. Thin layer chromatography was performed on
M-N ALUGRAM (registered) silica gel/ UV 254 sheets,
and detection of spots was made by UV light and/or iodine
vapors. Column chromatography was performed using
silica gel 70 230 mesh.
1
UV: λmax 240, 268, 276. HNMR (300 MHz, CDCl3) δ
ppm: 0.85 (3H, t, J = 6.3Hz, H-14′), 0.90 (3H, s, H-19),
1.18 (3H, s, H-20), 1.230 (28H, Br s, H-7, 16, 17, 4′–13′),
1.570 (2H, m, H-3′), 1.700 (2H, m, H-6), 1.95 (1H, t, J =
5.7 Hz, H-5), 2.12 (2H, t, J = 7.2 Hz, H-2′), 2.29 (2H, t, J =
7.5 Hz, H-1), 2.68 (2H, m, H-3), 2.86 (1H, septate, J = 6.6
Hz, H- 15), 3.12 (1H, dd, J = 6.6 and 13.8 Hz, H-18a), 3.22
(1H, dd, J = 6.6 and 13.8 Hz, H-18b), 7.28 (1H, d, J = 8.4
Hz, H-12), 7.39 (1H, d, J = 8.1 Hz, H-11), 7.82 (1H, s, H-
14). 13CNMR (100 MHz, CDCl3) δ ppm: 14.1 (C-14′), 18.5
(C-2), 18.6 (C-19), 18.8 (C-6), 22.6 (C-13′), 23.9 (C-17),
23.9 (C-16), 25.2 (C-20), 25.8 (C-3′), 29.6–29.1 (C-4′-11′),
30.1 (C-7), 31.8 (C-12′), 33.3 (C-15), 36.1 (C-3), 37.0 (C-
2′), 37.2 (C-4), 37.3 (C-10), 38.3 (C-1), 45.3 (C-5), 49.6 (C-
18), 123.7 (C-12), 124.0 (C-11), 126.8 (C-14), 134.7 (C-8),
145.5 (C-13), 147.0 (C-9), 173.2 (C-1′).
General procedure for the synthesis of amide derivatives
2–13
For the synthesis of amide carboxylic acid (1–2 mM) was
dissolved in toluene (10 ml) then solution of borane tetra-
hydrofuran (THF) (1 M) was added at 0 oC. After addition
of borane THF complex reaction mixture is kept at room
temperature for 1 h. DAA was added to the reaction mixture
and reaction mixture refluxed for 5–10 h. Then reaction
mixture was cooled to room temperature and acidifies with
1 M HCl, and extracted with ethyl acetate. Wash with water
2–3 times. Organic phase was dried over anhydrous Na2SO4
and concentrate under reduced pressure. Purification of
product was accomplished by column chromatography
using silica gel (hexane: ethyl acetate).
(+)-N-(palmitoyl)-dehydroabietylamine (4)
Rf: 0.686(hexane/ethylacetate, 9:1) State: gummy, Yield:
75.96%, [α]D26: −4.035 (C = 0.00545, CHCl3), HRMS-
ESI: calcd for C36H62NO (M+H)+ 524.4831, found
524.4808, IR (cm-1): 3303 (N–H stretching), 2923 (C–H),
2854 (C–H), 1646 (C=O), 1548 (N–H bending), 1460 (CH2
bending), 1373 (CH3 bending), 719 (CH2 long chain band).
(+)-N-(dodecanoyl)-dehydroabietylamine (2)
1
Rf: 0.627(hexane/ethylacetate, 9:1) State: gummy, Yield:
93.15%, [α]D26: −18.42 (C=0.0403, CHCl3), HRMS-ESI:
calcd for C32H54NO (M+H)+ 468.4205, found 468.4179,
IR (cm-1): 3303 (N–H stretching), 2923 (C–H), 2856(C–H),
1647 (C=O), 1548 (N–H bending), 1460 (CH2 bending),
1375 (CH3 bending), 719 (CH2 long chain band). UV: λmax
UV: λmax 240, 268, 276. HNMR (400 MHz, CDCl3) δ
ppm: 0.87 (3H, t, J = 6.4 Hz, H-16′), 0.98 (3H, s, H-19),
1.19 (3H, s, H-20), 1.20 (32H, Br s, H-7, 16, 17, 4′–15′),
1.609 (3H, m, H-15 and 3′), 2.13 (2H, t, J = 7.2 Hz, H-2′),
2.33 (2H, t, J = 7.6 Hz, H-1), 2.68 (2H, t, J = 4.8 Hz, H-3),
3.12 (1H, dd, J = 6.8 and 13.6 Hz, H-18a), 3.28 (1H, dd, J
= 6.4 and 13.6 Hz, H-18b), 7.15 (1H, d, J = 8.4 Hz, H-12),
7.28 (1H, d, J = 8.4 Hz, H-11), 7.84 (1H, s, H-14).
13CNMR (100 MHz, CDCl3) δ ppm: 14.0 (C-16′), 18.5 (C-
2), 18.7 (C-19), 18.9 (C-6), 22.6 (C-15′), 23.9 (C-17), 23.9
(C-16), 25.2 (C-20), 25.8 (C-3′), 29.3–29.6 (C-4′-14′), 30.1
(C-7), 31.8 (C-15), 35.9 (C-3), 36.9 (C-2′), 37.2 (C-4), 37.4
(C-10), 38.3 (C-1), 44.6 (C-5), 49.7 (C-18), 123.8 (C-12),
124.1 (C-11), 126.9 (C-14), 134.7 (C-8), 145.6 (C-13),
147.1 (C-9), 175.4 (C-1′).
1
241, 254, 276. HNMR (400 MHz, CDCl3) δ ppm: 0.86
(3H, t, J = 6.0 Hz, H-12′), 0.98 (3H, s, H-19), 1.23 (24H, Br
s, H-16, and 17, 3′–11′), 1.95 (1H, t, J = 5.6 Hz, H-5), 2.32
(4H, t, J = 7.6 Hz, H-1, and 2′), 2.86 (2H, m, H-3), 2.98
(1H, m, H-15), 3.17 (1H, dd, J = 6.4 and 13.6 Hz, H-18a),
3.28 (1H, dd, J = 6.4 and 13.6 Hz, H-18b), 7.28 (1H, d, J =
8.0 Hz, H-12), 7.39 (1H, d, J = 8.0 Hz, H-11), 7.83 (1H, s,
H-14). 13CNMR (100 MHz, CDCl3) δ ppm: 14.1 (C-12′),
18.5 (C-2), 18.7 (C-19), 18.9 (C-6), 22.6 (C-11′), 24.7 (C-
16 and 17), 25.2 (C-20), 29.5–29.3 (C-3′-9′), 31.8 (C-10′),
33.5 (C-15), 33.7 (C-2′), 35.7 (C-3), 37.3 (C-4), 37.4 (C-
10), 38.3 (C-1), 44.5 (C-5), 49.7 (C-18), 123.6 (C-12),
124.9 (C-11), 132.6 (C-14), 134.7 (C-8), 145.6 (C-13),
147.1 (C-9), 173.5 (C-1′).
(+)-N-(stearoyl)-dehydroabietylamine (5)
Rf: 0.647(hexane/ethylacetate, 9:1) State: gummy, Yield:
77.68%, [α]D26: −14.54 (C = 0.037, CHCl3), HRMS-ESI:

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Med Chem Res (2017) 26:1367–1376
calcd for C38H66NO (M+H)+ 552.5144, found 552.5174, IR
(cm-1): 3305 (N–H stretching), 2923 (C-H), 2852 (C-H),
1647 (C=O), 1550 (N–H bending), 1461 (CH2 bending),
1377 (CH3 bending), 721 (CH2 long chain band). UV: λmax
-17′), 1.56 (2H, m, H-3′), 1.71 (2H, m, H-6), 1.95 (4H, m,
H-8′ and 11′), 2.13 (2H, m, H-1), 2.67 (2H, m, H-2′), 2.97
(3H, m, H-15, and 7), 3.15 (1H, dd, J = 6.6 and 13.8 Hz, H-
18a), 3.28 (1H, dd, J = 6.6 and 13.8 Hz, H-18b), 5.30 (2H,
dt, J = 10.8 and 4.8 Hz, H-9′ and 10′), 7.13 (1H, s, H-14),
7.27 (1H, d, J = 8.4 Hz, H-12), 7.38 (1H, d, J = 8.4 Hz, H-
11). 13CNMR (100 MHz, CDCl3) δ ppm: 14.1 (C-18′), 18.1
(C-2), 18.5 (C-19), 18.9 (C-6), 22.6 (C-17′), 23.9 (C-17),
23.9 (C-16), 25.2 (C-20), 25.7 (C-3′), 27.1 (C-8′), 27.1 (C-
11′), 29.1 (C-5′), 29.2 (C-6′), 29.3 (C-7′), 29.4 (C-13′), 29.6
(C-14′), 29.7 (C-12′), 30.1 (C-7), 31.8 (C-16′), 33.5 (C-15),
35.9 (C-3), 36.1 (C-2′), 37.2 (C-4), 37.4 (C-10), 37.4 (C-1),
44.4 (C-5), 48.9 (C-18), 123.8 (C-12), 124.9 (C-11), 126.8
(C-14), 129.9 (C-9′), 132.6 (C-10′), 134.6 (C-8), 145.5 (C-
13), 147.1 (C-9), 173.5 (C-1′).
1
241, 269, 276. HNMR (600 MHz, CDCl3) δ ppm: 0.87
(3H, t, J = 6.6 Hz, H-18′), 0.99 (3H, s, H-19), 1.23 (37H, Br
s, H-16, 17, 20, 4′–17′), 1.48 (2H, t, J = 6.0 Hz, H-7), 1.62
(2H, m, H- 3′), 1.94 (1H, dd, J = 4.8, 12.6 Hz, H-5), 2.32
(2H, t, J = 7.2 and 15.0 Hz, H-2′), 2.68 (2H, m, H-3), 2.97
(1H, septate, J = 7.2 Hz, H-15), 3.15 (1H, dd, J = 6.6 and
13.8 Hz, H-18a), 3.28 (1H, dd, J = 6.6 and 13.8 Hz, H-18b),
7.27 (1H, d, J = 8.4 Hz, H-12), 7.38 (1H, d, J = 8.4 Hz, H-
11), 7.83 (1H, s, H-14). 13CNMR (100 MHz, CDCl3) δ ppm:
14.1 (C-18′), 18.5 (C-2), 18.7 (C-19), 18.9 (C-6), 22.6 (C-
17′), 23.9 (C-17), 23.96 (C-16), 24.7 (C-3′), 25.2 (C-20),
29.0–29.6 (C-4′-15′), 30.2 (C-7), 31.9 (C-16′), 33.4 (C-15),
33.8 (C-2′), 36.2 (C-3), 37.3 (C-4), 37.7 (C-10), 38.3 (C-1),
45.4 (C-5), 49.7 (C-18), 123.8 (C-12), 124.1 (C-11), 126.9
(C-14), 134.7 (C-8), 145.6 (C-13), 146.8 (C-9), 178.1 (C-1′).
(+)-N-(13-ene-docosanoyl)-dehydroabietylamine (8)
Rf: 0.568(Hexane/Ethylacetate, 9:1) State: gummy, Yield:
98%, [α]D26: −13.08 (C = 0.118, CHCl3), HRMS-ESI:
calcd for C42H72NO (M+H)+ 606.5614, found 606.5587,
IR (cm-1): 3304 (N–H stretching), 2925 (C–H), 2854
(C–H), 1646 (C=O), 1552 (N–H bending), 1460 (CH2
bending), 1379 (CH3 bending), 720 (CH2 long chain band).
(+)-N-(2-ene-butanoyl)-dehydroabietylamine (6)
Rf: 0.9019(chloroform/methanol, 9.5:0.5) State: gummy,
Yield: 100%, [α]D26: –10.12(C=0.0118, CHCl3), HRMS-
ESI: calcd for C24H36NO (M+H)+ 354.2797, found
354.2632, IR (cm-1): 3298 (N–H stretching), 2927 (C-H),
2868 (C-H), 1631 (C=O), 1548 (N–H bending), 1444 (CH2
bending), 1379 (CH3 bending). UV: λmax 242 1HNMR
(400 MHz, CDCl3) δ ppm: 0.92 (3H, s, H-19), 1.20 (9H, s,
H-16, 17, and 20), 1.42 (5H, m, H-1, 3, and 5), 1.80 (5H, m,
H-6 and 4′), 1.88 (2H, dd, J = 8.4, 6.8, 5.2 Hz, H-2), 2.27
(2H, m, H-1), 2.79 (1H, m, H-15), 2.87 (2H, J = 6.4 Hz, H-
7), 3.15 (1H, dd, J = 6.6 and 13.8 Hz, H-18a), 3.29 (1H, dd,
J = 6.6 and 13.8 Hz, H-18b), 5.76 (1H, d, J = 15.2 Hz, H-
2′), 6.81 (1H, d, J = 15.2 Hz, H-3′), 6.86 (1H, s, H-14), 6.97
(1H, d, J = 8.0 Hz, H-12), 7.15 (1H, d, J = 8.4 Hz, H-11).
13CNMR (100 MHz, CDCl3) δ ppm: 17.6 (C-4′), 18.5
(C-6), 18.7 (C-19), 18.9 (C-2), 23.9 (C-16 and 17), 25.2
(C-20), 30.2 (C-7), 33.4 (C-15), 36.2 (C-3), 37.4 (C-4), 37.5
(C-10), 38.3 (C-1), 45.2 (C-5), 49.6 (C-18), 122.3
(C-2′), 123.8 (C-12), 124.1 (C-11), 126.9 (C-14), 134.7 (C-
8), 139.8 (C-3′), 145.6 (C-13), 147.1 (C-9), 169.9 (C-1′).
1
UV: λmax HNMR (400 MHz, CDCl3) δ ppm: 0.86 (3H, t,
J = 6.4 Hz, H-22′), 0.91 (3H, s, H-19), 1.19 (3H, s, H-20),
1.25 (30H, Br s, H-16, 17, 4′–11′, 16′ -19′), 1.40 (2H, m, H-
3), 1.70 (1H, m, H-5), 1.73 (6H, m, H-2, 6, and 3′), 2.01
(4H, m, H-12′, 15′), 2.18 (2H, t, J = 7.6 Hz, H-2′), 2.83 (5H,
septate, J = 7.2 Hz, H-1, 7, and 15), 3.12 (1H, dd, J = 6.4
and 13.6 Hz, H-18a), 3.22 (1H, dd, J = 6.0 and 13.6 Hz, H-
18b), 5.34 (2H, dt, J = 10.8 and 4.8 Hz, H-13′ and 14′), 6.87
(1H, s, H-14), 6.97 (1H, d, J = 8.0 Hz, H-12), 7.15 (1H, d,
J = 8.4 Hz, H-11). 13CNMR (100 MHz, CDCl3) δ ppm:
14.0 (C-22′), 18.5 (C-2), 18.6 (C-19), 18.9 (C-6), 22.6 (C-
21′), 23.9 (C-17), 23.9 (C-16), 25.2 (C-20), 25.8 (C-3′),
27.1 (C-12′ and 15′), 29.2–29.7 (C-4′-11′ and 16′–19′), 30.1
(C-7), 31.8 (C-20′), 33.3 (C-15), 34.7 (C-8), 36.2 (C-3),
37.0 (C-2′), 37.2 (C-4), 37.4 (C-10), 38.3 (C-1), 45.3 (C-5),
49.6 (C-18), 123.8 (C-12), 124.0 (C-11), 126.8 (C-14),
129.8 (C-13′ and 14′), 134.7 (C-8), 145.5 (C-13), 147.1
(C-9), 173.1 (C-1′).
(+)-N-(oleanoyl)-dehydroabietylamine (7)
(+)-N-(9,12-diene-octadecanoyl)-dehydroabietylamine (9)
Rf: 0.637(Hexane/Ethylacetate, 9:1) State: gummy, Yield:
85.32%, [α]D26: –7.53 (C = 0.117, CHCl3), HRMS-ESI:
calcd for C38H64NO (M+H)+ 550.4988, found 550.4731,
IR (cm-1): 3304 (N–H stretching), 2925 (C–H), 2855 (C-H),
1646 (C=O), 1552 (N–H bending), 1459 (CH2 bending),
1379 (CH3 bending). UV: λmax 241, 268, 276. HNMR
(400 MHz, CDCl3) δ ppm: 0.86 (3H, t, J = 6.3 Hz, H-18′),
0.98 (3H, s, H-19), 1.22 (27H, Br s, H-16, 17, 20, 4′–7′, 12′
Rf: 0.617(Hexane/Ethylacetate, 9:1) State: gummy, Yield:
83.28%, [α]D26: −13.71 (C = 0.0060, CHCl3), HRMS-ESI:
calcd for C38H62NO (M+H)+ 548.4831, found 548.2537,
IR (cm-1): 3303 (N–H stretching), 2926 (C–H), 2858
(C–H), 1649 (C=O), 1550 (N–H bending), 1456 (CH2
bending), 1378 (CH3 bending), 720 (CH2 long chain band).
1
1
UV: λmax HNMR (400 MHz, CDCl3) δ ppm: 0.86 (3H,
brs, H-18′), 0.93 (3H, s, H-19), 1.21 (6H, d, J = 7.2 Hz, H-

Med Chem Res (2017) 26:1367–1376
1371
16 and 17), 1.27 (25H, Br s, H-7, 20, 3′–8′, 14′ -17′), 1.59
(2H, brs, H-2), 2.02 (1H, dd, J = 7.2 and 14.0 Hz, H-5),
2.15 (2H, t, J = 7.2 Hz, H-3), 2.32 (2H, t, J = 7.2 Hz, H-1),
2.77 (1H, septate, J = 6.4 Hz, H- 15), 2.89 (2H, dd, J = 6.0
Hz, H-7), 3.07 (1H, dd, J = 6.0 and 13.8 Hz, H-18a), 3.15
(1H, dd, J = 6.4 and 13.8 Hz, H-18b), 5.34 (4H, dt, J = 10.8
and 4.8 Hz, H-9′, 10′,12′ and 13′), 6.86 (1H, s, H-14), 6.97
(1H, d, J = 8.0 Hz, H-12), 7.14 (1H, d, J = 8.0 Hz, H-11).
13CNMR (100 MHz, CDCl3) δ ppm: 14.0 (C-18′), 18.5
(C-2), 18.7 (C-19), 18.9 (C-6), 22.5 (C-17′), 23.9 (C-17),
23.9 (C-16), 25.2 (C-20), 25.6 (C-3′), 27.1 (C-8′ and 14′),
29.0–29.6 (C-4′-7′ and 15′), 30.1 (C-7), 31.5 (C-16′), 33.5
(C-15), 37.2 (C-4), 37.4 (C-10), 37.5 (C-3), 38.3 (C-1), 45.4
(C-5), 49.7 (C-18), 123.8 (C-12), 124.1 (C-11), 126.9
(C-14), 127.8 (C-13′), 128.0 (C-12′), 130.0 (C-10′), 130.2
(C-9′), 134.7 (C-8), 145.6 (C-13), 147.1 (C-9), 173.2
(C-1′).
16 and 17), 1.58 (1H, m, H-5), 2.84 (1H, septate, J = 6.9
and 14.4 Hz, H-15), 3.07 (1H, dd, J = 6.6 and 13.8 Hz, H-
18a), 3.28 (1H, dd, J = 6.6 and 13.8 Hz, H-18b), 3.57 (2H,
s, H-2′), 6.83 (1H, s, H-14), 6.97 (2H, d, J = 8.4 Hz, H-11
and 12), 7.13 (1H, t, J = 7.6 Hz H-6′), 7.15 (2H, d, J = 7.6
Hz, H-4′ and 8′), 7.16 (2H, d, J = 7.6 Hz, H-5′ and 7′).
13CNMR (100 MHz, CDCl3) δ ppm: 18.4 (C-6), 18.5 (C-
19), 18.7 (C-2), 23.9 (C-17), 24.0 (C-16), 25.9 (C-7), 25.0
(C-20), 33.3 (C-15), 35.9 (C-3), 37.2 (C-4), 37.4 (C-10),
38.3 (C-1), 43.8 (C-2′), 44.9 (C-5), 60.3 (C-18), 123.6 (C-
12), 124.0 (C-11), 126.7 (C-14), 127.2 (C-6′), 128.9 (C-4′
and 8′), 129.2 (C-5′ and 7′), 134.5 (C-3′), 134.9 (C-8), 145.4
(C-13), 146.9 (C-9), 171.0 (C-1′).
(+)-N-(4(1H-indol-3-yl)-butanoyl)-dehydroabietylamine (12)
Rf: 0.882(chloroform/methanol, 9.5:0.5) State: gummy,
Yield: 99.45%, [α]D26: −13.26 (C = 0.0148, CHCl3),
HRMS-ESI: calcd for C32H43N2O (M+H)+ 471.3375,
found 471.2962, IR (cm-1): 3411 and 3303 (N–H
stretching), 2925 (C–H), 2866 (C–H), 1654 (C=O), 1525
(N–H bending), 1456 (CH2 bending), 1379 (CH3 bend-
ing). UV: λmax 240, 276, 291. 1HNMR (400 MHz,
CDCl3) δ ppm: 0.91 (3H, s, H-19), 1.19 (3H, s, H-20),
1.23 (6H, d, J = 6.8 Hz H-16 and 17), 1.25 (2H, m, H-3),
1.40 (1H, m, H-5), 1.85 (2H, m, H-2), 2.03 (2H, m, H-4′),
2.25 (4H, m, H-1 and2′), 2.84 (3H, septate, J = 7.2 Hz, H-
15 and 3′), 2.88 (2H, d, J = 6.4 Hz, H-7), 3.12 (1H, dd, J
= 6.8 and 14.0 Hz, H-18a), 3.19 (1H, dd, J = 6.0 and 13.6
Hz, H-18b), 6.85 (1H, s, H-6′) 6.89 (1H, s, H-14), 6.94
(1H, d, J = 8.0 Hz, H-10′), 7.01 (1H, d, J = 8.0 Hz, H-11),
7.13 (1H, t, J = 8.0 Hz, H-9′), 7.17 (1H, d, J = 8.0 Hz, H-
12), 7.31 (1H, d, J = 8.0 Hz, H-8′), 7.56 (1H, d, J = 7.6
Hz, H-11′). 13CNMR (100 MHz, CDCl3) δ ppm: 18.5 (C-
2), 18.7 (C-19), 18.9 (C-6), 23.9 (C-16), 24.0 (C-17), 24.5
(C-3′), 25.2 (C-20), 26.2 (C-4′), 30.1 (C-7), 33.4 (C-15),
35.8 (C-3), 36.3 (C-2′), 37.3 (C-4), 37.4 (C-10), 38.3 (C-
1), 45.2 (C-5), 49.6 (C-18), 111.1 (C-8′), 115.4 (C-5′),
118.8 (C-11′), 119.1 (C-9′), 121.5 (C-10′), 121.8 (C-6′),
123.8 (C-12), 124.1 (C-11), 126.9 (C-14), 127.4 (C-12′),
136.3 (C-7′), 134.8 (C-8), 145.6 (C-13), 147.2 (C-9),
173.1 (C-1′).
(+)-N-(benzoyl)-dehydroabietylamine (10)
Rf: 0.941(chloroform/methanol, 9.5:0.5) State: gummy,
Yield: 86.24%, [α]D26: –15.51 (C = 0.0486, CHCl3),
HRMS-ESI: calcd for C27H36NO (M+H)+ 390.2797, found
390.2487, IR (cm-1): 3334 (N–H stretching), 2925 (C–H),
2868 (C–H), 1641 (C=O), 1541 (N–H bending), 1459 (CH2
bending), 1380 (CH3 bending). UV: λmax 242 1HNMR
(400 MHz, CDCl3) δ ppm: 0.99 (3H, s, H-19), 1.19 (3H, s,
H-20), 1.22 (6H, d, J = 6.8 Hz, H-16 and 17), 1.37 (2H, d,
J = 13.2 Hz, H-3), 1.52 (1H, t, J = 13.2 Hz, H-5), 1.77 (2H,
m, H-2), 2.30 (2H, d, J = 12.8, H-1), 2.80 (1H, septate, J =
6.8 Hz, H-15), 2.90 (2H, d, J = 6.4 Hz, H-7), 3.36 (1H, dd,
J = 6.8 and 14.0 Hz, H-18a), 3.42 (1H, dd, J = 7.2 and 13.6
Hz, H-18b), 6.86 (1H, s, H-14), 6.98 (1H, d, J = 8.0 Hz, H-
12), 7.16 (1H, d, J = 8.4 Hz, H-11), 7.60 (1H, t, J = 7.6 Hz,
H-5′), 7.72 (2H, d, J = 7.6 Hz, H-4′ and H-6′), 8.10 (2H, d,
J = 7.6 Hz, H-3′ and 7′). 13CNMR (100 MHz, CDCl3) δ
ppm: 18.6 (C-2), 18.7 (C-19), 19.1 (C-6), 23.9 (C-16), 23.9
(C-17), 25.4 (C-20), 30.4 (C-7), 33.4 (C-15), 36.4 (C-3),
37.5 (C-4), 37.6 (C-10), 38.3 (C-1), 45.9 (C-5), 50.3 (C-18),
123.8 (C-12), 124.1 (C-11), 126.9 (C-14), 128.5 (C-4′),
131.3 (C-3′), 133.5 (C-5′), 134.7 (C-8), 134.8 (C-2′), 145.6
(C-13), 147.0 (C-9), 171.0 (C-1′).
(+)-N-(2-phenyl-acetyl)-dehydroabietylamine (11)
(+)-N-(2-(1H-indol-3-yl)-acetyl)-dehydroabietylamine (13)
Rf: 0.892(chloroform/methanol, 9.5:0.5) State: gummy,
Rf: 0.784(chloroform/methanol, 9.5:0.5) State: gummy,
Yield: 99%, [α]D26: −20.33(C=0.0151, CHCl3), HRMS-
ESI: calcd for C30H39N2O (M+H)+ 443.3062, found
443.2594, IR (cm-1): 3408 and 3284 (N–H stretching), 2925
(C–H), 2869 (C–H), 1652 (C=O), 1531 (N–H bending),
1456 (CH2 bending), 1388 (CH3 bending). UV: λmax 241,
274, 290. 1HNMR (400 MHz, CDCl3) δ ppm: 0.77 (3H, s, H-
19), 1.08 (3H, s, H-20), 1.26 (6H, d, J = 6.6 Hz H-16 and
26
Yield: 99.68%, [α]D
: −22.79 (C = 0.084, CHCl3),
HRMS-ESI: calcd for C28H38NO (M+H)+ 404.2953, found
404.2488, IR (cm−1): 3305 (N–H stretching), 2927 (C–H),
2869 (C–H), 1649 (C=O), 1552 (N–H bending), 1452 (CH2
bending), 1379 (CH3 bending). UV: λmax 240, 260, 276.
1HNMR (400 MHz, CDCl3) δ ppm: 0.82 (3H, s, H-19), 1.10
(2H, m, H-3),1.13 (3H, s, H-20), 1.23 (6H, d, J = 6.9 Hz, H-

1372
Med Chem Res (2017) 26:1367–1376
Scheme 1 Synthesis of amide
derivatives of DAA
17), 1.45 (1H, m, H-5), 2.87 (2H, d, J = 6.4 Hz, H-7), 2.90
(1H, m, H-15), 3.08 (1H, dd, J = 6.6 and 13.8 Hz, H-18a),
3.28 (1H, dd, J = 6.6 and 13.8 Hz, H-18b), 3.74 (2H, m,
H-2′), 6.82 (1H, s, H-14), 6.70 (1H, d, J = 8.4 Hz, H-11),
7.04 (1H, s, H-10′), 7.07 (1H, d, J = 8.4 Hz, H-12), 7.13 (1H,
m, H-7′), 7.19 (1H, J = 7.2 Hz, H-6′), 7.36 (1H, d, J = 7.8
Hz, H-8′), 7.54 (1H, d, J = 7.8 Hz, H- 5′). 13CNMR (100
MHz, CDCl3) δ ppm: 18.4 (C-2), 18.7 (C-6), 18.7 (C-19),
24.0 (C-17), 24.0 (C-16), 25.1 (C-20), 29.9 (C- 7), 33.4 (C-
15), 33.5 (C-2′), 35.8 (C-3), 37.2 (C-4), 37.3
(C-10), 38.1 (C-1), 44.5 (C-5), 49.3 (C-18), 109.1 (C-3′),
111.3 (C-8′), 118.4 (C- 5′), 120.1 (C-7′), 122.5 (C-6′), 123.5
(C-10′), 123.6 (C-12), 124.2 (C-11), 126.6 (C-4′), 126.7
(C-14), 134.7 (C-8), 136.3 (C-9′), 145.3 (C-13), 147.0
(C-9), 171.5 (C-1′).
in 90 to 100 % yield. Different spectroscopic techniques,
1
electrospray ionisation mass spectrometry, infrared, H-
nuclear magnetic resonance and 13C-nuclear magnetic
resonance spectroscopy ere used for characterization of
compounds.
Biological evaluation
Primary screening
In this study, a total of 12 novel acid amide derivatives
were screened for their cytotoxic effect on one breast
cancer and four HCC cell line by SRB technique as
described in experimental part. Trial drugs (2–13) were
introduced in two different (50 µM and 20 µM) con-
centrations in triplicate for each sample. Based on primary
screening results (Fig. 1a), 11 with a functional site of
phenyl acetic acid was found to be the most potent with
100% growth attenuation at 50 µM concentration on Huh7
and MCF7 cell line.
Results and discussion
Chemistry
Therefore, most potent trial drug 11 was further sub-
jected for systematic primary screening on ten HCC and one
breast cancer cell lines to determine inhibition rate of cells
at 10 µM concentration (Fig. 1b).
For the formation of amide bond, synthesis of active
intermediate is most important step. Recently, Huang
et al. (2007) reported an environmentally benign and
inexpensive method for the synthesis of amides that
avoids complicated purification procedures too (Yoon
et al. 1973). The synthesis of compounds 2–13 was
accomplished according to Scheme 1 using Zhongping
protocol a number of different acid amides were prepared
IC50 determination
As per above mentioned findings, five HCC including
Huh7, PLC/PRF/5, Snu475, Mahlavu and Hep3B-TR; and

Med Chem Res (2017) 26:1367–1376
1373
Fig. 1 a Cytotoxic SRB
screening results of 12
Primary Drug Screening
A
Huh7 at 20µM
Huh7 at 50µM
derivatives (2–13) against Huh7
and MCF7 cell lines at 50 and
20 Um. b Response of amide 11
at 10 µM concentration on cell
lines (n = 11)
3
9
8
12
4
5
8
3
5
4
9
2
2
7
13
10
6
7
11
10
6
12
11
13
0
20
40
60
80
100
120
0
20
40
60
80
100
Inhibition rate (%)
Inhibition rate (%)
5
4
8
3
9
2
7
10
12
6
MCF7 at 20µM
MCF7 at 50µM
5
4
3
9
8
2
12
10
7
6
11
13
13
11
0
20
40
60
80
100 120
0
20
40
60
80
100
Inhibition rate (%)
Inhibition rate (%)
B
Table 1 IC50 [µM] for compound 11 against six cell lines
triplicates ranging from 10 to 0.313 µM; for controls up to
0.1% DMSO final concentration was maintained with
respective media for each cell line; camptothecin as a
positive drug indicator was also used in triplicate at 5 µM
concentration and it showed 95–100% growth attenuation
on all cell lines used in this study.
As presented in Table 1, lowest IC50 value of 11 on
MCF7 (4.8 µM) was recorded. Moderate inhibitory
response on HCC cell lines was observed ranging from
PLC/PRF/5 (7.4 µM) to Mahlavu with the IC50 value of
39.7 µM, elucidating that amide 11 may have comparatively
higher therapeutic index on MCF7 cell line and HCC cell
lines than other trial drugs of the study.
Cell line
Cells/well Average control OD IC50 (µM) R2
MCF7
2500
1.0275
1.3472
0.5038
0.9402
0.914
4.8
7.4
1.0
1.0
1.0
0.8
1.0
0.8
PLC/PRF/5 5000
SNU475
Hep3B-TR
Huh7
2000
3500
2000
2000
9.8
11.7
11.8
39.7
Mahlavu
0.6662
breast cancer MCF7 cell lines were cultured in 96 well plate
in specified cell number/well for each cell line. After 24 h,
cells were treated with 11 at different concentrations in

1374
Med Chem Res (2017) 26:1367–1376
Comparison of cytotoxic effects of amide 11 with parent
DAA compound
We compared the cytotoxic activity of derivative 11 with
parent compound DAA on three cell lines, MCF7, PLC/
PRF/5 and Huh7. Three different concentrations were
applied, 10, 5 and 2,5 µM. At 10 µM parent compound
showed almost 100% growth attenuation, while at 5 µM
moderate inhibitory response of both 11 and DAA was
determined. Significant difference in rate of inhibition was
observed at 2.5 µM concentration between DAA and 11,
attributed to addition of N-phenacyl group at C-18 (Fig. 2).
Morphology
Reference to SRB findings, PLC/PRF/5 cell line was treated
for 72 h with 11 on 10 cm2 Petri plates. Photographs were
taken without fixation and cover slips were taken out to fix
Fig. 2 Inhibition rates of parent DAA and amide 11 at 5 µM and
2.5 µM concentration after 72 h
Fig. 3 a Photographs using 20X
A
lens, light microscope of PLC/
PRF/5 at 10 µM after 72 h of
administration of amide 11 after
fixation. b PLC/PRF/5:10 µM of
trial drug (11) after 48 and 72 h
before fixation
B
72 hrs Control
48 hrs Control
72 hrs (10µM, D-11)
48 hrs (10µMD-11)

Med Chem Res (2017) 26:1367–1376
1375
one breast cancer (MCF7) cell line. On comparison, sig-
nificant difference in growth attenuation rate was recorded
at 2.5 µM concentration of parent compound DAA and
phenacyl derivative 11. Low cell confluence and noticeable
increase in G2/M phase was recorded after 48 and 72 h of
treatment with 11 in PLC/PRF/5 cell line. Current findings
enlightened that amide 11 has comparatively sufficient
therapeutic significance and a potential candidate to be
further probed for future cancer interference and drug
research.
Acknowledgements One of the authors is thankful to Higher Edu-
cation Commission of Pakistan for providing fellowship. Muhammad
Ayaz Mustufa was supported by a TUBITAK (Turkey) PhD fellow-
ship. This work was partly supported by grants to M.O. from TUBI-
TAK
(111T558)
and
TUBA
(Turkish
Academy
of
Sciences). Conflict of Interest: The authors declared that they have
no competing interests.
Fig. 4 Cell proliferation comparison of DMSO control and trial drug
(11) of PLC/PRF/5 cell line after 48 h, showing changes in G2/M
phase
Compliance with Ethical Standards
Conflict of interest The authors declare that they have no competing
interests.
and stained with SRB dye. Using light microscope differ-
ences in cell morphology were observed in drug treated,
DMSO control and untreated cells and photographs were
taken at 20X after 72 h. Noticable morphological changes
resulting from low confluency, loss of cellular attachment
with clellular segregation and rounding of cells were
observed after 72 h among compound 11 treated cell line
(Figs 3a, b).
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