Synthesis, screening and pro-apoptotic activity of novel acyl spermidine derivatives on human cancer cell lines

Article abstract of DOI:10.1016/j.biopha.2017.06.019

The polyamines putrescine, spermidine, and spermine are polycationic, alkyl polyamines which play a significant role in eukaryotic cell proliferation. The polyamine metabolism and function are dysregulated in tumor cells making them an attractive therapeutic target by employing polyamine analogs. These analogs have a high degree of similarity with the structure of polyamines but not with their function. Multidrug resistance is a major factor in the failure of many chemotherapeutic drugs which necessitates further research and exploration of better novel alternatives. In the present study, Twenty-six novel acylspermidine derivatives were synthesized and evaluated for their anti-proliferative and pro-apoptotic activities on human breast cancer cells and T-lymphoblastic leukemia cells. The cell proliferation and apoptosis assays using WST-1 and annexin-V/7AAD staining respectively suggest that Compound 1 (C₁₉H₄₁N₃O₂) Compound 7(C₂₅H₅₁N₃O₂) and Compound 8 (C₂₉H₅₉N₃O) significantly reduced cancer cell viability in a dose- and time-dependent manner. Interestingly, compounds 7, 8 and 9 had slight or no effect on cell proliferation of non-cancerous cells. These studies speculate that these novel acylspermidine derivatives could be promising candidates in designing an anti-proliferative drug, targeting both solid and blood cancer cells.

Full text of DOI:10.1016/j.biopha.2017.06.019

Biomedicine & Pharmacotherapy 93 (2017) 190–201
Available online at
ScienceDirect
www.sciencedirect.com
Original article
Synthesis, screening and pro-apoptotic activity of novel acyl
spermidine derivatives on human cancer cell lines
Syed Shoeb Razvia, Hany Choudhrya,b,c,e, Said Salama Moselhya,d,e,f
,
Taha Abduallah Kumosania,d,g, Mohammed Nihal Hasana, Mazin A. Zamzamia,b,c
,
Khalid Omer Abualnajaa,d,e, Abdulrahman Labeed Al-Malkia,d,e, Mahmoud Alhosin
a,b,c,1,**
, Tadao Asamia,h,1,
*
a
Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia
Cancer Metabolism and Epigenetic Unit, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia
Cancer and Mutagenesis Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia
Experimental Biochemistry Unit, King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia
b
c
d
e
Bioactive Natural Products Research Group, King Abdulaziz University, Jeddah, Saudi Arabia
f
Biochemistry Department, Faculty of Science, Ain shams University, Cairo, Egypt
g
Production of Bioproducts for Industrial Applications Research Group, King Abdulaziz University, Jeddah, Saudi Arabia
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo, 113-8657, Japan
h
A R T I C L E I N F O
A B S T R A C T
Article history:
The polyamines putrescine, spermidine, and spermine are polycationic, alkyl polyamines which play a
significant role in eukaryotic cell proliferation. The polyamine metabolism and function are dysregulated
in tumor cells making them an attractive therapeutic target by employing polyamine analogs. These
analogs have a high degree of similarity with the structure of polyamines but not with their function.
Multidrug resistance is a major factor in the failure of many chemotherapeutic drugs which necessitates
further research and exploration of better novel alternatives. In the present study, Twenty-six novel
acylspermidine derivatives were synthesized and evaluated for their anti-proliferative and pro-apoptotic
activities on human breast cancer cells and T-lymphoblastic leukemia cells. The cell proliferation and
apoptosis assays using WST-1 and annexin-V/7AAD staining respectively suggest that Compound 1
(C19H41N3O2), Compound 7(C25H51N3O2) and Compound 8 (C29H59N3O) significantly reduced cancer cell
viability in a dose- and time-dependent manner. Interestingly, compounds 7, 8 and 9 had slight or no
effect on cell proliferation of non-cancerous cells. These studies speculate that these novel
acylspermidine derivatives could be promising candidates in designing an anti-proliferative drug,
targeting both solid and blood cancer cells.
Received 12 March 2017
Received in revised form 4 June 2017
Accepted 5 June 2017
Keywords:
Acylspermidines
Polyamines
Cancer therapy
Apoptosis
© 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction
therapy but with the due course of time and treatment, cells
acquire resistance through mutation or downregulation of the pro-
Cancer still remains the most common cause of death
worldwide with the gradual increase of the incidences with age.
Though initially, it responds to the chemotherapy or the radiation
apoptotic proteins and the overexpression of anti-apoptotic
proteins such as bcl-2 [1,2]. Carcinogenesis involves dysregulation
of the complex processes like apoptosis, angiogenesis and cell
proliferation. Usage of phytochemicals or the synthetic analogs of
the metabolites involved in the cell proliferation is on the rise for
the treatment of the cancerous lesions triggering apoptosis. Many
anticancer chemotherapeutic drugs have been developed which
induce apoptosis [3,4].
Polyamines are positively charged molecules which have a
crucial role in the proliferation and normal growth of a cell [5].
They are known for their unique interaction with a variety of
biological targets, mostly with membrane phospholipids and
* Corresponding author at: Graduate School of Agricultural and Life Sciences, The
University of Tokyo, Bunkyo, Tokyo, 113-8657, Japan.
** Corresponding author at: Department of Biochemistry, Cancer Metabolism and
Epigenetic Unit, Faculty of Sciences, King Abdulaziz University, P.O. Box 80203,
Jeddah 21589, Saudi Arabia.
E-mail addresses: malhaseen@kau.edu.sa (M. Alhosin), brassinazole@gmail.com
(T. Asami).
1
Equal contributors.
http://dx.doi.org/10.1016/j.biopha.2017.06.019
0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.

S.S. Razvi et al. / Biomedicine & Pharmacotherapy 93 (2017) 190–201
191
nucleic acids through the electrostatic interactions completing the
2. Material and methods
protonation of amino groups at physiological pH [6]. Spermine,
spermidine, and putrescine are the natural polyamines studied
widely due to the various receptors, ion channels, and the other
recognition sites to which they bind [7]. Many ligands have been
designed with different and specific biological targets by insertion
of suitable pharmacophores onto the backbone of polymethylene
including the adjustment of the position of the amine which can
optimize the selectivity and affinity of the receptor [2,5,8].
During the recent years, many compounds of polyamine
backbone have been synthesized which demonstrated anti-
proliferative effect against diverse malignant tumors. Synthetic
polyamine analogs like bisnaphthalimido or N1, N11-diethyl
norspermine against breast cancer are significant with regard to
anti-neoplastic activities against many solid tumor models of
humans [9,10].
Many of the anticancer drugs that are widely clinically used
today have high risks of adverse side effects and poor specificity
and selectivity against tumors. Hence, there arises an urgent need
to encounter this problem by improving the efficacy, selectiveness,
and safety of the anti-tumor agents which can be done by
modification with specific polyamine motifs [7] elevating the
affinity for tumor cells and reach the biological targets. Therefore,
we synthesized novel conjugates of acyl polyamines with fatty acyl
group as a primary polyamine motif on the polyamine backbone. It
has been hypothesized by many researchers that breast cancer is
one of the most chemo-sensitive solid tumors, most of the initially
responsive tumors regress and then develop resistance to a broad
spectrum of drugs [11,12].
Moreover, the human breast cancer cell line MCF-7 is considered
as the most common malignant type of breast cancer used widely
around the laboratories as a tumor model in screening to study the
anticancer effects and also in the investigation of the mechanism of
action of hormones [13–15]. Additionally, the T-lymphoblastic
leukemia cell line Jurkat is also a documented experimental model
for hematological cancers in the screening of the anticancer activity
aswellas the cellularandmolecularmechanismsofdrugsexhibiting
anticancer proprieties [16–18].
The aim of this study was to evaluate the anti-proliferative and
pro-apoptotic effects of synthetic acylspermidines on the human
adenocarcinoma cell line MCF-7 and the T-lymphoblastic leukemia
cell line Jurkat.
2.1. The general scheme of synthesis of active compounds
The general scheme of synthesis of the compounds is depicted
in Fig. 1.
2.2. Synthesis of N4-(3-hydroxylauroyl) spermidine (compound 1)
2.2.1. N1,N8-bis-tert-butoxycarbonylspermidine
2-(tert-Butoxycarbonyloxyimino)-2-phenylacetonitrile (4.85 g,
19.7 mmol) in dry THF (25 mL) was added dropwise to a solution of
spermidine (1.36 g, 9.36 mmol) and triethylamine (3.91 mL,
28.1 mmol) in dry THF (25 mL) with cooling in an ice bath and
then stirred overnight at room temperature. After the reaction, the
solvent was removed in vacuo, and 1 M NaOH (50 mL) was added to
the resultant residue. The aqueous layers were extracted with
dichloromethane (50 mL Â 3). The combined organic layers were
washed with saturated aqueous sodium chloride (50 mL), dried
over Na2SO4 and concentrated in vacuo to give white solid, which
was then recrystallized with n-hexane/dichloromethane to give
white solid of N1,N8-bis-tert-butoxycarbonylspermidine (2.58 g,
7.47 mmol, 79.8%).
1H NMR (500 MHz, CDCl3)
d 5.13 (s, 1H, NHCO), 4.80 (s, 1H,
NHCO), 3.27–3.15 (m, 2H, CH2NHCO), 3.14–3.02 (m, 2H, CH2NHCO),
2.65 (t, J = 6.6 Hz, 2H, (CH2)4NHCH2), 2.59 (t, J = 6.6 Hz, 2H,
(CH2)3NHCH2), 1.72–1.58 (m, 2H, NHCH2CH2CH2NHCO), 1.58–
1.46 (m, 4H, NHCH2CH2CH2CH2NHCO), 1.42 (s, 18H, C(CH3)3).
2.2.2. N1, N8-bis-tert-butoxycarbonyl-N4-(3-hydroxylauroyl)
spermidines
Under nitrogen atmosphere, 1-(3-Dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (665 mg, 3.47 mmol) was added
to a solution of N1, N8-bis-tert-butoxycarbonylspermidine (1.00 g,
2.89 mmol), 3-hydroxylauric acid (626 mg, 2.89 mmol) and dime-
thylamino pyridine (424 mg, 3.47 mmol) in dichloromethane
(20 mL), and stirred for 2 days at room temperature. Then a
solution of 10% (w/w) citric acid (10 mL) was added to the reaction
Fig 1. Overview of synthesis of active compounds. Compound 1: (R = -CH2-CH(OH)-(CH2)7-CH3); Compound 7: (R = -CH2-(CH2)5-CH2-CH = CH-CH2-CH(OH)-(CH2)5-CH3);
Compound 8: (R = -CH2-(CH2)9-CH2-CH = CH-CH2-(CH2)7-CH3); Compound 9:(R = -CH2-(CH2)6-CH2-CH = CH-CH3).

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S.S. Razvi et al. / Biomedicine & Pharmacotherapy 93 (2017) 190–201
mixture and stirred for 10 min. The reaction mixture was extracted
with dichloromethane (10 mL Â 3), dried over Na2SO4 and concen-
trated in vacuo. The residue was purified by silica gel column
chromatography (n-hexane: ethyl acetate = 1:1) to give N1, N8-bis-
tert-butoxycarbonyl-N4-(3-hydroxylauroyl) spermidine as a color-
less oil (1.03 g, 1.91 mmol, 66.1%), which was sufficiently pure for
further use.
stirred for 10 min. The reaction mixture was extracted with
dichloromethane (3 mL Â 3), dried over Na2SO4 and concentrated
in vacuo. The residue was purified by silica gel column
chromatography (n-hexane: ethyl acetate = 1:1) to give N1, N8-
bis-tert-butoxycarbonyl-N4-(erucoyl)
spermidine
(297 mg,
0.447 mmol, 89.4%) as a colorless oil, which was sufficiently pure
for further use.
2.2.3. N4-(3-hydroxylauroyl) spermidine
2.4.2. N4-(erucoyl)spermidine
Under nitrogen atmosphere, N1, N8-bis-tert-butoxycarbonyl-
N4- (3-hydroxylauroyl) spermidine (950 mg, 1.74 mmol) was
dissolved into 50% (v/v) trifluoroacetic acid solution in dichloro-
methane (10 mL) and stirred for 20 min. After the addition of
methanol (30 mL), the solvent was removed in vacuo. Then 1 M
NaOH (10 mL) was added, and the aqueous solution was extracted
with dichloromethane (10 mL Â 3). The combined organic layers
were dried over Na2SO4 and concentrated in vacuo to give N4-(3-
hydroxylauroyl) spermidine as a colorless oil (535 mg, 1.56 mmol,
54.0%).
Under nitrogen atmosphere, N1, N8-bis-Boc-N4-(erucoyl) sper-
midine (297 mg, 0.447 mmol) was dissolved into 50% (v/v)
trifluoroacetic acid solution in dichloromethane (3 mL) and stirred
for 20 min. After the addition of methanol (10 mL), the solvent was
removed in vacuo. Then 1 M NaOH (3 mL) was added, and the
aqueous solution was extracted with dichloromethane (3 mL Â 3).
The combined organic layers were dried over Na2SO4 and
concentrated in vacuo to give N4-(erucoyl) spermidine as a pale-
yellow oil (204 mg, 0.437 mmol, 97.7%).
2.5. N4-(10-undecenoyl) spermidine (compound 9)
2.3. Synthesis of N4-(ricinoleoyl) spermidine (compound 7)
2.5.1. N1, N8-bis-tert-butoxycarbonyl-N4-(10-undecenoyl)
spermidines
2.3.1. N1, N8-bis-tert-butoxycarbonyl-N4-(ricinoleoyl) spermidines
Under nitrogen atmosphere, 1-(3-Dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (230 mg, 1.20 mmol) was added
to a solution of N1, N8-bis-tert-butoxycarbonylspermidine (346 mg,
1.00 mmol), ricinoleic acid (299 mg, 1.00 mmol) and dimethyla-
mino pyridine (146 mg,1.20 mmol) in dichloromethane (6 mL), and
stirred for 2 days at room temperature. Then a solution of 10% (w/
w) citric acid (3 mL) was added to the reaction mixture and stirred
for 10 min. The reaction mixture was extracted with dichloro-
methane (5 mL Â 3), dried over Na2SO4 and concentrated in vacuo.
The residue was purified by silica gel column chromatography (n-
hexane: ethyl acetate = 1:1) to give N1, N8-bis- tert-butoxy
carbonyl-N4-(ricinoleoyl) spermidine (198 mg, 0.316 mmol,
31.6%) as a colorless oil, which was sufficiently pure for further use.
Under nitrogen atmosphere, 1-(3-Dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (152 mg, 0.79 mmol) was added
to a solution of N1, N8-bis-tert-butoxycarbonylspermidine (228 mg,
0.662 mmol), 10-undecenoic acid (122 mg, 0.662 mmol) and
dimethylamino pyridine (96 mg, 0.79 mmol) in dichloromethane
(3 mL), and stirred for 2 days at room temperature. Then a solution
of 10% (w/w) citric acid (3 mL) was added to the reaction mixture
and stirred for 10 min. The reaction mixture was extracted with
dichloromethane (3 mL Â 3), dried over Na2SO4 and concentrated
in vacuo. The residue was purified by silica gel column
chromatography (n-hexane: ethyl acetate = 1:1) to give as a
colorless oil N1, N8-bis-tert-butoxycarbonyl-N4-(10-undecenoyl)
spermidine (302 mg, 0.590 mmol, 89.4%), which was sufficiently
pure for further use.
2.3.2. N4-(ricinoleoyl)spermidine
2.5.2. N4-(10-undecenoyl) spermidines
Under nitrogen atmosphere, N1, N8-bis-Boc-N4-(ricinoleoyl)
spermidine (198 mg, 0.316 mmol) was dissolved into 50% (v/v)
trifluoroacetic acid solution in dichloromethane (3 mL) and stirred
for 20 min. After the addition of methanol (30 mL), the solvent was
removed in vacuo. Then 1 M NaOH (3 mL) was added, and the
aqueous solution was extracted with dichloromethane (3 mL Â 3).
The combined organic layers were dried over Na2SO4 and
concentrated in vacuo to give N4-(ricinoleoyl) spermidine as a
pale-yellow oil (132 mg, 0.310 mmol, 98.1%).
Under nitrogen atmosphere, N1, N8-bis-Boc-N4-(10-undece-
noyl) spermidine (302 mg, 0.590 mmol) was dissolved into 50% (v/
v) trifluoroacetic acid solution in dichloromethane (3 mL) and
stirred for 20 min. After the addition of methanol (10 mL), the
solvent was removed in vacuo. Then 1 M NaOH (3 mL) was added,
and the aqueous solution was extracted with dichloromethane
(3 mL Â 3). The combined organic layers were dried over Na2SO4
and concentrated in vacuo to give N4-(10-undecenoyl) spermidine
as a pale-yellow oil (182 mg, 0.584 mmol, 99.9%).
2.4. Synthesis of N4-(erucoyl) spermidine (compound 8)
2.6. Preparation of stock solution and dilution
2.4.1. N1, N8-bis-tert-butoxycarbonyl-N4-(erucoyl) spermidines
Under nitrogen atmosphere, 1-(3-Dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (115 mg, 0.60 mmol) was added
to a solution of N1, N8-bis-tert-butoxycarbonylspermidine (173 mg,
0.502 mmol), erucic acid (170 mg, 0.502 mmol) and dimethylamino
pyridine (73 mg, 0.60 mmol) in dichloromethane (3 mL), and
stirred for 2 days at room temperature. Then a solution of 10%
(w/w) citric acid (3 mL) was added to the reaction mixture and
The Stock solutions of the drugs with 100 mM concentration
were prepared in DMSO (SIGMA-ALDRICH, USA, catalog no. D8418)
for compound 7 and 8. While the remaining compounds were
prepared in tissue culture grade sterile water (InvitrogenTM USA,
catalog no. 15230-204) and stored at À20 ꢀC. Working standard
dilutions to 1 mM were done in tissue culture grade sterile water
and kept at 4 ꢀC. For each experiment, fresh dilution of the drug

S.S. Razvi et al. / Biomedicine & Pharmacotherapy 93 (2017) 190–201
193
Table 1
Molecular structures and formulae of acylspermidine derivatives.
Compound
Molecular formula
Structure
1
C19H41N3O2
C19H41N3O2
C19H41N3O2
2
3
4
C19H41N3O2
5
C19H41N3O2
6
C19H41N3O2
7
8
C25H51N3O2
C29H59N3O
9
C18H37N3O

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S.S. Razvi et al. / Biomedicine & Pharmacotherapy 93 (2017) 190–201
Table 1 (Continued)
Compound
Molecular formula
Structure
10
11
C17H37N3O2
C19H41N3O2
12
C19H41N3O2
13
14
C19H41N3O2
C19H41N3O2
15
16
17
C25H51N3O
C25H53N3O
C19H39N3O2
18
19
C17H35N3O4
C23H38N4O8S

S.S. Razvi et al. / Biomedicine & Pharmacotherapy 93 (2017) 190–201
195
Table 1 (Continued)
Compound
Molecular formula
Structure
20
21
C13H24Cl2N4O4S
C10H15N3O4S
22
23
C19H21N3O5S
C19H21N3O6S
24
25
C25H35N3O6SSi
C18H28BrNO2Si
26
C18H29NO3Si
was used in treatment and for all the experiments where DMSO
was used as a solvent, the concentration of DMSO was maintained
at 0.1- 0.5%.
2.8. Cell viability assay
The effect of the acylspermidine derivatives on proliferation/
inhibition was analyzed by a colorimetric cell proliferation assay
using WST-1 reagent (Sigma-Aldrich, USA, catalog no.
11644807001). Briefly, the cells were seeded in 96-multiwell
plates at a density of 104 cells/well (counted using Scepter 2.0
Handheld Automated Cell Counter, Millipore1, Billerica, MA,
catalog no. PHCC20040) for MCF-7, fibroblasts or 4 Â104/well for
Jurkat cells. After 24 h of incubation, the cells were exposed to
2.7. Cell culture and treatment
The human breast adenocarcinoma cell line (MCF-7) and
human lymphocytic leukemia cell line (Jurkat) were obtained from
the American Type Culture Collection (ATCC, Manassas, VA, USA)
and maintained in a humidified incubator with 5% CO2 at 37 ꢀC.
MCF-7 cells were grown in Dulbecco’s Modified Eagle’s Medium
(DMEM) (UFC biotech, Riyadh, KSA, catalog no. T-DMEM-111014)
and Jurkat cells were cultured in RPMI 1640 (UFC biotech, Riyadh,
KSA, catalog no. TRPMI-11111). Both DMEM and RPMI were
supplemented with 10% FBS (Lifetech, catalog no. 16000-044) and
1% penicillin-streptomycin antibiotics (100 units/mL, Lifetech,
catalog no. 15140-122). Normal human dermal fibroblasts (NHDFs)
(catalog no. PCS-201-012) were procured from the ATCC and grown
in fibroblast basal media (ATCC, catalog no. PCS 201-030)
supplemented with fibroblast low kit growth serum (ATCC, catalog
no. PCS 201-041) and penicillin-streptomycin antibiotics (100
units/mL, Lifetech, catalog no. 15140-122).
different concentrations (5, 10, 30 and 50 mM) of acyl spermidines
for the desired time periods. Cell proliferation rate was evaluated
through a rapid WST-1 reagent. After incubation for the above-
mentioned time duration, 10 mL of the WST-1 solution was added
and incubated for an additional 4 h at 37 ꢀC. Finally, the absorbance
was read at 450 nm with a microplate ELISA reader (ELx800TM
Biotek, USA) and the results were analyzed by the Gen5 software
(Biotek, USA). Thymoquinone (Sigma-Aldrich, USA, catalog no.
274666-5G) was used as a positive control. The percentage of cell
viability was calculated by assuming control (untreated) samples
as 100% viable [19]. The IC50 (50% inhibitory concentration) values
were calculated from the dose-response curves.

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S.S. Razvi et al. / Biomedicine & Pharmacotherapy 93 (2017) 190–201
Fig. 2. Compounds 1, 7, 8 and 9 reduces the cell viability in a concentration-dependent way in cancer cells. MCF-7 and Jurkat cells were treated with several concentrations of
compound 1 (A), compound 7 (B), compound 8 (C) and compound 9 (D) for 24 h. Cell viability rate was assessed using WST-1 assay. Represented values are mean Æ S.E.M. of
three experiments (n = 3); statistically significant (unpaired ‘t’ test, two-tailed): *, p < 0.05; **, p < 0.01; (versus the corresponding untreated group).
Benchtop Flow Cytometer (Millipore1, Billerica, MA), and the
Table 2
results plotted using the InCyteTM software (Millipore1, Billerica,
MA).
IC50 values (mM) of the active compounds against tumor cells are presented as
mean Æ S.E.M.
Compound
MCF-7
JURKAT
2.10. Statistical analysis
1
7
8
9
35.37 Æ 2.1
21.25 Æ 2.5
23.22 Æ 1.7
22.41 Æ 5.5
16.75 Æ 6.3
56.71 Æ 5.4
61.78 Æ 4.1
33.17 Æ 0.7
56.29 Æ 1.0
18.91 Æ 3.7
All the data were presented as Mean Æ SEM (standard error of
the mean) of triplicates done in the same experiment or an average
of at least three independent experiments. The differences
between the control and the treated groups for cell proliferation
and apoptosis were analyzed by Student’s t-test (two-tailed) using
GraphPad Prism 6 (Graph Pad Software, SanDiego, USA) and the
significant differences were indicated as *P < 0.05, **P < 0.001,
***P < 0.0001 (Table 2).
Thymoquinone
2.9. Apoptosis assay
MCF7 (2 Â105 cells/well in 6 well plate) and Jurkat cells (4 Â104
cells/well in 96 well plate) were cultured overnight and treated
with different concentrations of the tested compounds. After
treating the cells with desired concentration of the drugs for
several time periods, Annexin V/7AAD staining was carried out as
3. Results
3.1. NMR data of the active compounds
per the manufacturer’s protocol. Briefly, 100 mL of the nexin
reagent (Millipore1, Billerica, MA, catalog no. 4500-0450) staining
solution (containing annexin V-fluorescein and 7AAD) was added
and incubated at room temperature for 20 min in the dark
surroundings. The forward and side scatter were recorded at
10,000 events and the subsequent percentage of the early, and the
late apoptotic cells were analyzed using the Guava1 easyCyte 12HT
Compound 1: (Yield 54%) 1H NMR (500 MHz, CDCl3) d 3.99 (m,
1H, CH(OH)), 3.46–3.25 (m, 4H, CH2NCH2), 2.76–2.54 (m, 4H,
2 Â CH2NH2), 2.50–2.32 (m, 2H,COCH2), 1.70–1.50 (m, 6H,
NCH2CH2CH2CH2NH2, NCH2CH2CH2NH2), 1.45 (m, 2H, CH(OH)
CH2CH2), 1.26 (m, 14H, (CH2)7CH3), 0.88 (t, J = 7.0 Hz, 3H, CH3).

S.S. Razvi et al. / Biomedicine & Pharmacotherapy 93 (2017) 190–201
197
Fig. 3. Compounds 1, 7, 8 and 9 reduces cell viability in a time-dependent manner in cancer cells. MCF-7 and Jurkat cells were treated with 50
m
M of the compound 1 (A),
compound 7 (B), compound 8 (C) and compound 9 (D) for several time durations. Cell viability rate was assessed using WST-1 assay. Values are mean Æ S.E.M. of three
experiments (n = 3); statistically significant (unpaired ‘t’ test, two-tailed): *, p < 0.05; **, p < 0.01; ***, p < 0.001 (versus untreated group).
Fig. 4. Compounds 1, 7, 8 and 9 exhibit slight or no cytotoxicity on normal Fibroblasts. NHDFs (normal human dermal fibroblasts) were treated with several concentrations of
compound 1, compound 7, compound 8 and compound 9 for 24 h. Cell viability rate was assessed using WST-1 assay. Represented values are mean Æ S.E.M. of three
experiments (n = 3); statistically significant (unpaired ‘test, two-tailed): *, p < 0.05 (versus the corresponding untreated group).
Compound 7: (Yield 98.1%) 1H NMR (500 MHz, CDCl3)
d
5.63–
CH2CH = CHCH2), 1.86–1.40 (m, 8H, NCH2CH2CH2CH2NH2,
5.33 (m, 2H, CH = CH), 3.71–3.50 (m, J = 6.0 Hz, 1H, CH(OH)), 3.50–
3.17 (m, 4H, CH2NCH2), 2.82–2.57 (m, 4H, 2 Â CH2NH2), 2.41–2.23
(m, 2H, COCH2), 2.20 (t, J = 6.6 Hz, 2H, CHCH2CH(OH)), 2.04 (q,
J = 6.9 Hz, 2H, CHCH2CH2),1.87–1.51 (m,10H, NCH2CH2CH2CH2NH2,
NCH2CH2CH2NH2, COCH2CH2 CH(OH)CH2CH2), 1.51–1.19 (m, 16H,
(CH2)4, (CH2)4), 0.88 (t, J = 6.9 Hz, 3H, CH3).
NCH2CH2CH2NH2,
COCH2CH2(CH2)8, CH3(CH2)6), 0.87 (t, J = 6.9 Hz, 3H, CH3).
Compound 9: (Yield 99.9%) 1H NMR (500 MHz, CDCl3)
(ddt, J = 20.1, 6.9, 6.3 Hz, 1H, CH = CH2), 4.96 (dd, J = 17.2, 1.7 Hz, 1H,
1/2CH = CH2), 4.90 (dd, J = 10.0, 1.4 Hz, 1H, 1/2CH = CH2), 3.47–3.16
(m, 4H, CH2NCH2), 2.79–2.59 (m, 4H, 2 Â CH2NH2), 2.35–2.20 (m,
2H, COCH2), 2.01 (q, J = 7.1 Hz, 2H, CH2CH = CH2), 1.73–1.48 (m, 8H,
NCH2CH2CH2CH2NH2, NCH2CH2CH2NH2, COCH2CH2),1.48–1.07 (m,
10H, (CH2)5).
COCH2CH2),
1.37–1.11
(m,
28H,
d
5.78
Compound 8: (Yield 97.7%) 1H NMR (500 MHz, CDCl3)
d 5.40–
5.29 (m, 2H, CH = CH), 3.49–3.15 (m, 4H, CH2NCH2), 2.80–2.60 (m,
4H, 2 Â CH2NH2), 2.35–2.22 (m, 2H, COCH2), 2.00 (q, J = 6.4 Hz, 4H,

198
S.S. Razvi et al. / Biomedicine & Pharmacotherapy 93 (2017) 190–201
Fig. 5. Compounds 1, 7 and 8 induce concentration-dependent apoptosis in Jurkat cells. Cells were exposed to increasing concentrations of compound 1 (A), compound 7 (B)
and compound 8 (C) for 24 h. Apoptosis in Jurkat cells was assessed by flow cytometry using the Annexin V/7AAD staining apoptosis assay (D). Values are Mean Æ S.E.M. of
three experiments (n = 3); statistically significant (unpaired ‘test, two-tailed): *, p < 0.05; ***, p < 0.001 (versus untreated group).
3.2. Compounds 1, 7, 8, and 9 decreased cell viability of cancer cells by
dose-dependent mechanism
3.3. The cell viability inhibition induced by compounds 1, 7, 8 and 9
was time-dependent
Initially, we screened the 26 derivatives of acyl spermidines on
MCF-7 cells to assess the anti-proliferative activity of the
compounds (Table 1). The findings have shown that compounds
1, 7, 8 and 9 significantly decreased MCF-7 cell viability (15%, 12%,
21% & 13% for 1, 7, 8 and 9 respectively at highest concentration) in
We next investigated the time-dependent effect of the
compounds 1, 7, 8 and 9 at a concentration of 50
time periods (3, 6 and 9 h). Incubation of MCF-7 cells with 50
m
M for different
M of
m
compounds 1, 7 and 8 induced a time-dependent decrease in cell
viability with a significant visual effect after 3 h (Fig. 3A–C).
However, the decline in the cell viability of MCF-7 after exposure to
compound 9, showed a significant effect at 9 h with a cell viability
of 55% (Fig. 3D). Compared to compounds 1, 7 and 9, compound 8
was more active against Jurkat cells in decreasing the cell viability
(Fig. 3). Indeed, Compound 9 decreased the cell viability of Jurkat
cells by about 60% and 40% after an incubation of 6 h and 9 h
respectively (Fig. 3D). Such effect was not observed in Jurkat cells
by the Compounds 1, 7 and 9 under similar conditions. Altogether,
a concentration-dependent (5, 10, 30 and 50 mM) manner (Fig. 2,
(Supplementary Figs. S1–S3 in the online version at DOI: 10.1016/j.
biopha.2017.06.019)). The corresponding IC50 values were
calculated as depicted in Table 2. The most active compound on
MCF-7 was Compound 7 with an IC50 of 21.25 Æ 2.5
compound 9, 8 and 1 with IC50 values of 22.41 Æ5.5, 23.22 Æ1.7 and
35.37 Æ2.1 M respectively (Fig. 2 and Table 2). Unlike MCF-7 cells,
compound 8 was the most active compound against Jurkat cells
with IC50 value of 33.17 Æ 0.7 M, followed by compound 9,1 and 7
mM followed by
m
m
these findings indicate that compounds 1,
7 and 8 target
with IC50 values of 56.29 Æ1.0, 56.71 Æ5.4 and 61.78 Æ 4.1
mM
predominantly MCF-7 cells relative to Jurkat cells and compound
8 could target both solid and blood tumors.
respectively. Thymoquinone was used as a positive control which
showed significant decrease in the cell viability with an IC50 value
of 16.75 Æ 6.3
m
M on MCF-7 and 18.91 Æ3.7
mM on Jurkat cells
3.4. Compounds 1, 7, 8 and 9 demonstrated minimal cytotoxicity
towards non-cancerous cells
respectively (Table 2).
Thereafter, compounds 1, 7, 8 and 9 were selected for further
cytotoxic studies on MCF-7 and Jurkat cells. Altogether, this data
indicates that compounds 1, 7 and 9 target predominantly MCF-7
cells relative to Jurkat cells and compound 8 could be a selective
inhibitor of cell proliferation of both MCF-7 cells and Jurkat cells.
To investigate the selectivity of the active compounds towards
the cancer cells, we treated the normal human dermal fibroblasts
(NHDFs) with compounds 1, 7, 8 and 9 under the similar
experimental conditions that were used for tumor cells (Fig. 4).

S.S. Razvi et al. / Biomedicine & Pharmacotherapy 93 (2017) 190–201
199
Fig. 6. Compound 8 induces a time-dependent apoptosis in Jurkat cells. Cells were treated with 50
m
M of compound 8 (A) for different time durations or with 30
m
M of
thymoquinone (B) for 24 h. (C) Apoptosis in Jurkat cells was assessed by flow cytometry using the Annexin V/7AAD staining apoptosis assay. Values are mean Æ S.E.M. of three
experiments (n = 3); statistically significant (unpaired ‘test, two-tailed): **, p < 0.01; ***, p < 0.001 (versus untreated group).
Although, compound 1, 7, 8 and 9 at a concentration of 50
significantly induced cell proliferation inhibition of MCF-7 cells by
about 80% (Fig. 2), no such effect was found in NHDFs (Fig. 4).
m
M,
compound 8 induced a time-dependent apoptosis (Fig. 6). A
significant increase in apoptotic cells was found already at 3 h
(34.5%) and was more prominent at 6 h and 9 h by about 68% and
However, at 50
m
M, compounds 1, 7, 8 and 9 induced slight
83.1% respectively. When Jurkat cells were incubated with 50 mM
inhibition of fibroblasts cell viability by about 23%, 13%, 8% and 15%
respectively (Fig. 4) suggesting that the compound 1 showed more
cytotoxicity relative to the other compounds towards NHDFs.
of compound 8, it induced a time-dependent increase in annexin
V-positive cells and this effect was quite significant commencing
from 3 h and reached higher levels to approximately 76% at 9 h
(Fig. 6A). Then, we analyzed the effect of compounds 1, 7, 8 on the
3.5. Compound 1, 7 and 8 induced apoptosis in MCF-7 & Jurkat cells
apoptosis in MCF-7 at a concentration of 50
mM (Fig. 7). Unlike
Jurkat cells, the compound was the most pro-apoptotic
1
To determine the compound-induced mechanism(s) involved
in cell viability inhibition, we studied their effect on the apoptosis
in both the cancer cell lines. Annexin V-7AAD staining was carried
out after treatment with either desired concentrations of com-
pounds 1, 7 and 8 for 24 h or with the natural anticancer
compound, thymoquinone as a positive control. Fig. 5 shows that
all the three compounds (1, 7 and 8) were able to induce significant
compound among the others (Fig. 7B). While compound 7 and 8
induced apoptosis in MCF-7 by about 23% and 18% respectively, the
percentage of apoptotic cells reached to 59% in compound 1-
treated MCF-7 cells (Fig. 7A and B). Altogether, these findings
suggest that MCF-7 and Jurkat cells respond in different ways to
compound 1, 7 and 8 suggesting that these compounds trigger
apoptosis in solid and hematological tumors by different signaling
pathways.
apoptosis in Jurkat cells, starting from 10
apoptotic cells was more pronounced with the higher concen-
trations (30 and 50 M). Treating Jurkat cells using 50 M of either
mM. The percentage of
m
m
4. Discussion
compound 1 (Fig. 5A) or 7 (Fig. 5B) induced apoptosis by about 12%
and 14% respectively. Interestingly, compound 8 was able to induce
apoptosis in Jurkat cells by more than 70% under the same
conditions (Fig. 5C). These findings indicate that compound 8 is the
most active pro-apoptotic compound in Jurkat cells compared to 1
and 7 which is in agreement with the findings of cell viability
(Fig. 2). To determine the chronology of the compound 8-induced
molecular and cellular events precisely, we studied its time course
The natural polyamines are involved in multiple cellular
functions. These polyamines have a pivotal role in cell growth
and proliferation which makes them a promising target for anti-
cancer therapy[1,20]. Thus, there is an increasing demand to
develop competitive polyamine analogs which compete for uptake
of natural polyamines and hence shutting down the biosynthesis of
polyamines in the cell by initiating polyamine degradation [21].
effects on Jurkat cells at a concentration of 50 mM. Interestingly,

200
S.S. Razvi et al. / Biomedicine & Pharmacotherapy 93 (2017) 190–201
Fig. 7. Compounds 1, 7 and 8 induces a concentration-dependent apoptosis in MCF-7cells. Cells were exposed to increasing concentrations of compound 1, compound 7 and
compound 8 for 24 h. (A) Apoptosis in MCF-7 cells was assessed by flow cytometry using the Annexin V/7AAD staining apoptosis assay and the percentage of annexin-V
positive cells are depicted (B). Values are mean Æ S.E.M. of three experiments (n = 3); statistically significant (unpaired ‘t’ test, two-tailed): *, p < 0.05; ***, p < 0.001 (versus
untreated group).
The present study indicates that Compound 1(C19H41N3O2),
Compound 7 (C25H51N3O2) and Compound 8 (C29H59N3O) signifi-
cantly reduced the cell viability of the human breast cancer cell line
MCF-7 by dose- and time-dependent mechanisms. Unexpectedly,
compounds 7 and 8 had no same effect on the apoptosis when
compared to cell viability indicating that these compounds can
trigger cell death mechanisms other than apoptosis. Other
mechanisms including autophagy have been shown to be involved
in the acylspermidine analog-induced cell proliferation inhibition
[22,23]. In agreement with this hypothesis, the GC7 (N1guanyl-1,7-
diamineoheptane), spermidine analog was shown to induce
autophagy in Human fibrosarcoma cells [22]. Compounds 1, 7
and 8 also reduced cell viability and induced apoptosis in Jurkat
cells in a dose- and time-dependent manner. However, the anti-
proliferative and pro-apoptotic activities were more pronounced
with compound 8 indicating that this derivative could be a potent
candidate which could target both solid and hematological tumors.
Moreover, This data shows that Compounds 1, 7 and 8 targets
predominantly MCF-7 relative to Jurkat cells. The present study is
in good agreement with the recent findings that the use of
polyamine analogues like N1, N11-diethylnorspermine (DENSPM)
[10] and bis-amino-oxy-propyl-naphthalimido polyamine deriva-
tives [9] against breast cancer cells and the use of hydroxylated
polyamine analogs against leukemia cells [24] could be an effective
measure to check the proliferation of cancer cells. Interestingly,
except compound 1, the other active compounds 7, 8 and 9 had no
or slight effect on cell viability of the NHDFs which were used as a
non-cancerous cell model in this study. These observations
support our conclusion that compound 8 could be a very promising
inhibitor of cancer cell proliferation by targeting polyamine
pathways without affecting the normal cells. Lipophilicity of the
compounds plays a significant role in the transport of polyamines
into the cell and the subsequent cytotoxic effect on the tumor cells.
The lipophilic nature of Compound 8 is rendered by the presence of
alkyl group on the side chain of the polyamine which makes it
more penetrable through the cytoplasmic membrane via endocy-
tosis. This speculation may explain its superior pro-apoptotic
effects on both the cancer lines.
During the recent years, many polyamine analogs have been
designed which initiate an apoptotic cascade of events in the
tumors of skin, colon, breast, liver and prostate [25,26].
Phosphatidylserine (PS) is an essential phospholipid present in
the inner leaflet of the plasma membrane in normal healthy
mammalian cells. PS is exposed to the exterior when the cell
membrane is compromised during cell death which is a hallmark
of early apoptosis. This exposed PS can be stained easily using
annexin V which specifically binds to it and can be detected by
Flowcytometric assays. Exposure of Jurkat cells to compound 8
induced a rapid increase in the annexin V positive cells. This effect
was quite significant commencing from 3 h indicating that
compound 8 can decrease the cell viability of cancer cells by
induction of apoptosis, but the involvement of other mechanisms
is not excluded. Our findings are quite similar to a previous study
wherein the bisnaphthalimido polyamines derivatives were
evaluated against tumor cells [9]. Cancer cells thrive and
proliferate unchecked in the polyamine-rich environments and

S.S. Razvi et al. / Biomedicine & Pharmacotherapy 93 (2017) 190–201
201
hence the induction of apoptosis can be the means to keep a check
on the growth by siphoning off the polyamines which are required
for cell growth and proliferation. Interestingly, in the present
study, Compound 8 has shown inhibitory effect on tumor cell
proliferation without any detrimental effect to the non-cancerous
cells.
In conclusion, the present study highlights the potential of
synthesized acyl spermidine derivatives in triggering apoptosis in
both solid and hematological tumors. This study shows that cancer
cells can respond to acylspermidine derivatives in different ways.
Interestingly, most of the active compounds had no or slight
inhibitory effects on normal cells. The present findings suggest that
compound 8 could be a selective trigger of cell death in cancer cells
and this effect could be attributed to its structure. Therefore,
further studies are required to determine the signaling pathways
involved in the apoptosis, induced by these synthesized deriva-
tives.
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Conflict of interest
[16] U. Schneider, H.U. Schwenk, G. Bornkamm, Characterization of EBV-genome
negative null and T cell lines derived from children with acute lymphoblastic
leukemia and leukemic transformed non-Hodgkin lymphoma, Int. J. Cancer.19
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The authors declare that they have no financial conflict of
interest.
[17] A. Fauster, M. Rebsamen, K.V.M. Huber, J.W. Bigenzahn, A. Stukalov, C.-H.
Lardeau, S. Scorzoni, M. Bruckner, M. Gridling, K. Parapatics, J. Colinge, K.L.
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Acknowledgements
This project was funded by the Deanship of Scientific Research
(DSR), at King Abdulaziz University, Jeddah, under grant no: 1-130-
36-HiCi. The authors acknowledge the efforts of DSR for technical
and financial support.
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