Synthesis, docking, cytotoxicity, and LTA4H inhibitory activity of new gingerol derivatives as potential colorectal cancer therapy

Article abstract of DOI:10.1016/j.bmc.2016.12.048

Leukotriene A4 hydrolase (LTA₄H) is a proinflammatory enzyme that generates the inflammatory mediator leukotriene which may play an important role in chronic inflammation associated carcinogenesis. [6]-gingerol, the major bioactive compound of Zingiber officinale, is a potential inhibitor of LTA₄H, a highly expressed enzyme in colorectal carcinoma. Eighteen compounds; seven of natural origin (including [4]-, [6]-, [8]-, and [10]-gingerol), five new and six known semi-synthesized [6]-gingerol derivatives were examined using docking, in vitro cytotoxicity against human colon cancer cells (HCT-116) and LTA₄H aminopeptidase and epoxide hydrolase inhibitory studies. Methyl shogoal (D8) showed to be the most potent compound against HCT-116 cells (IC₅₀; 1.54?μM). Remarkably, D8 proved to be non-cytotoxic to normal cells; (TIG-1) and (HF-19) with high selective index (SI; 52.3). Furthermore [6]-gingerol derivatives showed potent LTA₄H inhibitory activities in comparison to the universal positive controls (bestatin and 4BSA). Among the natural gingerols, [10]-gingerol (N3) exhibited the highest LTA₄H aminopeptidase and epoxide hydrolase inhibitory activities with IC₅₀; 21.59 and 15.24?μM, respectively. Meanwhile, methyl shogoal (D8) and 4′-O-prenyl-[6]-gingerol (D10) retained the highest inhibition with IC₅₀; 4.92 and 3.01?μM, for aminopeptidase, and 11.27 and 7.25?μM for epoxide hydrolase activities, respectively.

Full text of DOI:10.1016/j.bmc.2016.12.048

Accepted Manuscript
Synthesis, Docking, Cytotoxicity, and LTA4H Inhibitory Activity of New Gin-
gerol Derivatives as Potential Colorectal Cancer Therapy
Mai H. El-Naggar, Amira Mira, Fatma M. Abdel Bar, Kuniyoshi Shimizu,
Mohamed M. Amer, Farid A. Badria
PII:
S0968-0896(16)31149-X
http://dx.doi.org/10.1016/j.bmc.2016.12.048
BMC 13475
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
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Accepted Date:
7 November 2016
27 December 2016
Please cite this article as: El-Naggar, M.H., Mira, A., Abdel Bar, F.M., Shimizu, K., Amer, M.M., Badria, F.A.,
Synthesis, Docking, Cytotoxicity, and LTA4H Inhibitory Activity of New Gingerol Derivatives as Potential
Colorectal Cancer Therapy, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.
2016.12.048
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Synthesis, Docking, Cytotoxicity, and LTA4H Inhibitory Activity of
New Gingerol Derivatives as Potential Colorectal Cancer Therapy
Mai H. El-Naggara,b, Amira Miraa, Fatma M. Abdel Bara, Kuniyoshi Shimizuc, Mohamed M.
Amera, Farid A. Badriaa,*
Affiliation
a
Pharmacognosy Department, Faculty of Pharmacy, Mansoura University, Mansoura 35516
Egypt
b PharmacognosyDepartment, Faculty of Pharmacy, Sohag University, Sohag, Egypt
cDivision of Systematic Forest and Forest Products Sciences, Department of Agro-
Environmental Sciences, Faculty of Agriculture, Graduate School of Kyushu University, Japan
Mai Elnaggar; mai.h.elnaggar@gmail.com
Amira Mira: dramiramera@gmail.com
Fatma M. Abdel Bar: fatma_maar@yahoo.com
Kuniyoshi Shimizu: shimizukuni@gmail.com
Mohamed M. Amer; pharmamer_47@yahoo.com
Farid A. Badria; faridbadria@gmail.com
Correspondence: Farid A. Badria, Ph. D., Pharmacognosy Department, Mansoura University,
Mansoura 35516, Egypt.E-mail: faridbadria@gmail.com
Phone: +201001762927Fax: +20502247496
1

Abstract
Leukotriene A4 hydrolase (LTA4H) is a proinflammatory enzyme that generates the
inflammatory mediator leukotriene which may play an important role in chronic inflammation
associated carcinogenesis. [6]-gingerol, the major bioactive compound of Zingiber officinale, is a
potential inhibitor of LTA4H, a highly expressed enzyme in colorectal carcinoma. Eighteen
compounds; seven of natural origin (including [4]-, [6]-, [8]-, and [10]-gingerol), five new and
six known semi-synthesized [6]-gingerol derivatives were examined using docking, in vitro
cytotoxicity against human colon cancer cells (HCT-116) and LTA4H aminopeptidase and
epoxide hydrolase inhibitory studies. Methyl shogoal (D8) showed to be the most potent
compound against HCT-116 cells (IC50; 1.54 mM). Remarkably, D8 proved to be non-cytotoxic
to normal cells; (TIG-1) and (HF-19) with high selective index (SI; 52.3). Furthermore [6]-
gingerol derivatives showed potent LTA4H inhibitory activities in comparison to the universal
positive controls (bestatin and 4BSA). Among the natural gingerols, [10]-gingerol (N3) exhibited
the highest LTA4H aminopeptidase and epoxide hydrolase inhibitory activities with IC50; 21.59
and 15.24 mM, respectively. Meanwhile, methyl shogoal (D8) and 4`-O-prenyl-[6]-gingerol
(D10) retained the highest inhibition with IC50; 4.92 and 3.01 mM, for aminopeptidase, and 11.27
and 7.25 mM for epoxide hydrolase activities, respectively.
Key words
Zingiber officinale, Gingerols, Semisynthesis, HCT-116, LTA4H, aminopeptidase, epoxide
hydrolase
2

Introduction
Leukotriene A4 hydrolase (LTA4H) is a bifunctional zinc-dependent metalloenzyme with the
activities of epoxide hydrolase and aminopeptidase. As an epoxide hydrolase, LTA4H catalyzes
the final rate-limiting step in the biosynthesis of leukotriene B4 (LTB4), a potent chemo-
attractant that induces a vigorous inflammatory response and is implicated in cancer
development as depicted in Fig 1A.1, 2 The active site of LTA4H is composed of a wide open
central section containing catalytic zinc connected to a narrow and deeper L-shaped
hydrophobic cavity in which the hydrophobic tail of LTA4 is buried strongly, Fig. 1B.3
Compilation of information from biochemical studies, mutational analysis, and X-ray
crystallography leads to the conclusion that the two enzyme activities of LTA4H are exerted via
distinct and overlapping active sites. Thus, certain residues are specifically required for the
aminopeptidase reaction such as Glu-296 and Tyr-383, whereas, Asp-375 is critical only for the
epoxide hydrolase reaction. On the other hand, Glu-271, Arg-563, and the zinc atom are
necessary for both catalytic reactions. In fact, Glu-271 is a unique example of a residue that is
shared between two catalytic machineries, carrying out a separate chemistry in each of the two
enzyme reactions.3
Plant-derived chemoprevention and/or dietary phytochemicals have emerged as an available and
promising strategy towards cancer control and management with several mechanisms including
targeting LTA4H.4, 5 [6]-gingerol, the major compound of Zingiber officinale rhizomes and
proved to exhibit a wide array of biochemical and pharmacological activities.7- 9 [6]-gingerol was
also reported to play a role in cancer chemotherapy and chemoprevention.1, 10-12 Several studies
suggested that [6]-gingerol is effective in the suppression of mutagenesis, angiogenesis and
metastasis. 10-13 The possible mode of action of [6]-gingerol was postulated by Jeong et al. by
3

using reverse-docking, in vitro, and in vivo experiments to disclose that LTA4H might be a
potential target of [6]-gingerol. These results may explain the efficacy of [6]-gingerol in
targeting LTA4H, a highly expressed enzyme in colorectal carcinoma for the prevention of
colorectal cancer.1
Human colorectal-carcinoma is the third most common cancer diagnosed in both men and
women worldwide. 14Treatment options are limited with poor efficacy and marked patient-to-
patient variation in therapeutic outcomes. In most cases, surgical resection and organ
transplantation remain the only curative treatment options that may involve very expensive and
invasive procedures with considerable limitations. The current cornerstone of adjuvant and
palliative chemotherapy for colorectal cancer is 5-FU.15 Nevertheless, the clinical usefulness of
such compound is limited by its remarkable side effects such as myelotoxicity, gastrointestinal
disturbances, cardiotoxicity, and hepatotoxicity.16These limitations directed towards finding a
more effective and safe drug which may raise the therapeutic benefits for patients.
Despite the several lines of evidence supporting the activity of [6]-gingerol against
multiple human cancers, no thorough study to date has profiled the anti-colon cancer activity of
other gingerol derivatives. Furthermore, this study pursues the relationships between the
structure elements of these gingerol derivatives and their anti-colon cancer activities using two
complementary approaches; docking study and in vitro enzyme assay. This prompted us to
pursue a virtual screening (docking), in vitro cytotoxicity study using HCT-116 and two normal
cell lines; TIG-1 and HF-19, and LTA4H aminopeptidase and epoxide hydrolase inhibitory
assays for both natural and semisynthesized gingerol derivatives.
2. Results and discussion
2.1. Chemistry
4

Compounds (N1-N7), Fig. 2, were previously isolated by our group from Z. officinale Roscoe
dry rhizomes (Zingiberaceae)17 whereas [6]-gingerol (N1) was isolated in a high yield (0.06%
w/w)and was used in the current study for the preparation of gingerol derivatives (D1-D11), Fig.
2. Moreover,[8]-gingerol (N2); [10]-gingerol (N3); [4]-gingerol (N4); 4`-O-methyl-[6]-gingerol
(N5); a new gingerol derivative, namely, Δ8-[10]-gingerol(N6); and a known sesquiterpene
isolated for the first time from ginger namely 1,2-dihydroxybisabola-3,10-diene (N7) were used
along with the derivatives (D1-D11) in docking, in vitro cytotoxicity, and LTA4H inhibitory
activity experiments.
Williamson ether reaction of N1 with allyl bromide produced D1.The 1H-NMR data of
D1 (Table 1) revealed the close resemblance to that of N1 except for the presence of extra
signals accounted for an allyl group at d 5.96 (1H, m, H-9`), 5.30 (2H, d, J=17.6 Hz, 10`b), 5.19
(2H, d, J=10.4 Hz, H-10`a) and 4.50 (2H, d, J=5.6 Hz, H-8`). This was confirmed from13C-NMR
signals (Table 2) at d 132.8 (C-9`), 116.9 (C-10`), and 68.9 (C-8`). The downfield shift of the
aromatic carbons, C-3` (+2.0 ppm), C-4` (+1.4 ppm), and the upfield shift of C-5` (-3.4 ppm) in
comparison to those of [6]-gingerol indicated an expected allylation of the phenolic OH group of
[6]-gingerol. Thus, the structure of D1 was established to be a new gingerol derivative namely,
4`-O-allyl-[6]-gingerol.
Acetylation (Ac2O/pyridine) of N1 resulted in two products; a major one designated asD3
and a minor one designated as D2. The downfield shift of C-5 signal in D3 (+2.7 ppm) compared
to that of [6]-gingerol suggested acetylation of the alcoholic OH group at C-5. In addition, the
downfield shift of the aromatic carbons, C-3` (+4.5 ppm), C-5` (+8.2 ppm), and the upfield shift
of C-4` (-6.0 ppm) in comparison to those of [6]-gingerol indicated an expected acetylation of
the phenolic OH group. The presence of two acetyl groups was confirmed from the1H-NMR
5

singlets at d 2.23 and 1.92 (H-9` and H-12, respectively) and 13C-NMR signals at d 170.5 and
169.2(C-11 and C-8`, respectively) and at d 21.1 and20.7 (C-12 and C-9`, respectively). Thus,
D3 was established to be diacetyl derivative of [6]-gingerol. This compound was previously
reported, 18- 20 however, its spectroscopic data is not available in the literature. The minor
product was established to be 4`-acetyl-[6]-shogaol (D2) by comparison of its spectroscopic data
with that reported in literature.21
Methylation (CH3I/K2CO3) of N1 resulted in five products, D4-D8. The NMR spectral
data of D6 showed the presence of three methoxyl groups indicating two new methylation
positions. These methoxyl groups represented by 1H-NMR signals at d 3.21 (3H, s, H-11), 3.77
(3H, s, H-7`) and 3.79 (3H, s, H-8`) and confirmed by 13C-NMR signals at d 56.9, C-11; 55.9, C-
8` and 55.8, C-7`. The strong downfield shift of C-5 signals in D6 (+9.6 ppm) compared to that
of [6]-gingerol suggested methylation of the OH group at C-5. While, the downfield shift of the
aromatic carbons, C-3` (+2.5 ppm), C-4` (+3.4 ppm), and the upfield shift of C-5` (-3.1 ppm)
indicated methylation of the phenolic OH group. Accordingly, the structure of D6 was assigned
to be a new dimethylated derivative namely, 4`,5-O-dimethyl-[6]-gingerol.
Compound D5 was established to be 5-O-methyl-[6]-gingerol, which had been detected
in ginger by GC-MS before,22, 23 however, no previous reports for its spectral data. Therefore, we
reported herein the 1H and 13C-NMR data of this compound, Table 1 and 2.
The structures of the remaining products, D4,D7 and D8 were confirmed by comparison of their
spectral data with that reported in literature and they were found to be methyl-[6]-shogaol
(D8),21, 24 [6]-shogaol (D7),24- 26 and methyl zingerone (D4).27
6

Oxidation (Collins reagent) of N1 afforded D9.The NMR spectral data of D9 (Table 1
and 2) was very close to that of N1 except for the signals corresponding to the aromatic ring. The
1H-NMR data (Table 1) showed only one signal in the aromatic region at d 6.73, br s that is
equivalent to two protons of a tetra substituted aromatic ring. In addition, the 13C-NMR data
(Table 2) showed the disappearance of one methine signal (CH-5) of the aromatic ring and a
quaternary appeared instead. The previous findings and the strong downfield shift of C-5` in D9
(+10.0 ppm) accompanied by slight downfield shift of carbons C-4` and C-6` in comparison to
those of [6]-gingerol suggested dimerization of [6]-gingerol at C-5`. This was confirmed with
LC/MS/MS spectral data that showed [M-H]+ peak at m/z 585.4 corresponding to a molecular
formula of C34H49O8. Accordingly, the structure of D9 was established to be a new [6]-gingerol
dimer namely,bis-(I-5`→II-5`)-[6]-gingerol, that may be formed by oxidative phenol coupling
reaction.
Prenyl derivatives (D10 and D11) were prepared by using Williamson ether synthesis
(Prenyly bromide/ K2CO3) reaction of N1. The 1H-NMR data of the major one (D10, Table 1)
was very close to that of N1 except for the presence of extra signals of a prenyl moiety at d 4.55
(2H, d, J=6.6 Hz, H-8`), 5.51 (1H, t, J=6.5 Hz, H-9`), 1.74 (3H, s, 11`) and 1.78 (3H, s, 12`).
This was also confirmed fromits 13C-NMR data (Table 2) that showed signals of the prenyl
moiety at d (65.9, C-8`), (120.0, C-9`), (137.4, C-10`), (18.2, C-11`) and (25.8, C-12`). The
downfield shift of the aromatic carbons, C-3` (+3.1 ppm), C-4` (+2.7 ppm), and the upfield shift
of C-5` (-2.5 ppm) in comparison to those of [6]-gingerol (N1) indicated an expected prenylation
of the phenolic OH group. Thus, the structure of D10 was assigned to be a new gingerol
derivative namely, 4`-O-prenyl-[6]-gingerol. The NMR data of the minor product (D11) was
very close to that of D10 except for its 13C-NMR spectra (Table 2)that exhibited signals for two
7

olefinic carbons at d 147.8 (C-5) and 130.3 (C-4). Also, an upfield shift (-11.6 ppm) of its C-3to
d 199.8 compared to4`-O-prenyl-[6]-gingerolsuggesting the presence of an a,b-unsaturated
ketone. This was supported by the olefinic proton signals (Table 1) at d 6.10 (1H, br d, J=15.1
Hz, H-4) and 6.85 (1H, m, H-5) with trans configuration indicating a possible dehydration of
prenyl-[6]-gingerol into prenyl-[6]-shogaol. Consequently, compound D11 was established to be
a new [6]-gingerol derivative namely, 4`-O-prenyl-6-shagaol.
2.2. Molecular modeling and Docking study
In the present study, a computational virtual screening of [6]-gingerol and other gingerol
derivatives against LTA4H was carried out using PyRx 0.8 as virtual screening software,28 and
AutoDock Vina as a molecular docking program.29 It was found that [6]-gingerol does not share
exactly the same localization of bestatin within the LTA4H catalytic pocket as reported by Jeong
et al..1 However, it shares overlapping localization between bestatin (a classic LTA4H inhibitor)
and 4BS (4-amino-N-[4-(benzyloxy)-phenyl]butanamide), another LTA4H inhibitor, which is
cocrystallized with LTA4H in the crystal structure of PDB code: 3CHR, as shown in Figure 3A.
Furthermore, several structural characteristics for [6]-gingerol binding with LTA4H, were
identified.Firstly, [6]-gingerol had L-shaped backbone conformation similar to that of
leukotriene A4 (LTA4),the natural substrate of LTA4H, which fit the L-shaped hydrophobic
cavity in LTA4Hprotein (Figure 1A). Secondly, the phenolic OH group in [6]-gingerol was
found to be essential for inhibiting both LTA4H activities due to its coordination with zinc metal
and that may explain the inhibition of the aminopeptidase activity of the enzyme(Figure 3B).
Next, the OH group at C-5 of [6]-gingerol which has S-configuration forms a hydrogen bond
with Asp-375 which is essential for the epoxide hydrolase activity only and it was reported to be
responsible for stereospecific introduction of a hydroxyl group at C-12 of LTA4.30 These
8

resultsare consistent with the evidence emerging from Jeonget al. study that reported reduction in
the binding affinity of [6]-gingerol to [D375A] LTA4H mutant form.1 Finally, the carbonyl
oxygen of the ligand forms a hydrogen bondwith Gln-134.
In silico docking calculations, performed with AutoDock Vina (Table 3), identified Δ8-[10]-
gingerol (N6) with an unsaturation center as having the highest binding affinity to LTA4H (-9.5
Kcal/mol) among the tested gingerol derivatives (Figure 3C). Additionally, the gingerol
homologues (N2-N4) which differ only in the length of the aliphatic side chain that is buried into
the hydrophobic pocket of the protein (Figure 3D) had close binding affinities to that of N1
towards LTA4H (-.8.8 to -9.3 Kcal/mol). However, [10]-gingerol N3, showed the highest score
amongst these series. Substitution of the alcoholic hydroxyl group in [6]-gingerol decreased the
binding affinity to LTA4H due to losing the hydrogen bond interaction with the carboxyl side
chain of Asp-375 as shown in 5-O-methyl-[6]-gingerol (D5), Figure 4A.However, losing of the
alcoholic OH group through dehydration in shogaol (D7) enhanced the binding affinity due to
the formation of a,b-unsaturated ketone with a conjugated system that mimics the conjugated
triene in the natural substrate, LTA4 (Figure 4B). Allylation of the phenolic group of [6]-
gingerol was shown to disrupt the coordination with zinc metal in allyl-[6]-gingerol (D1), Figure
4C, Likewise, saturation of the aromatic ring in N7decreasedthe binding affinity to LTA4H (-7.4
Kcal/mol).Dimerization of [6]-gingerol, as in D9 led to dramatic decrease in the binding affinity
to LTA4H (-7.7 Kcal/mol) due to bulkiness and steric hindrance (Figure 4D).
2.3. Cytotoxicity Assay
The cytotoxic activity of gingerol derivatives against human colon cancer cells (HCT-116) was
evaluated, and compared to that of bestatin and 4BSA as positive controls (Table 4). Noticeably,
the shogoal derivative D8, showed potent cytotoxicity against HCT-116 cells (IC50;1.54 mM) in
9

comparison to the positive controls, bestatin (IC50; 42.54 mM) and 4BSA (IC50; 30.54 mM).
Furthermore, D8 showed very high selectivity index 'SI" equals 52.3. Additionally, the
semisynthetic derivatives, D7, D10, D2 and D3 (IC50; 12.87, 21.51, 26.87 and 30.95 mM,
respectively) showed higher activity than the parent compound N1 (IC50 45.05 mM) and also
showed selectivity index 'SI" > 3, indicating less cytotoxicity to normal cells.
The tested derivatives were non-cytotoxic to the normal cells; TIG-1 and HF-19 and showed IC50
range from 54.8 to100 mM that indicated their selectivity in targeting colon cancer cells.
2.4. Aminopeptidase and epoxide hydrolase inhibitory activity
Aminopeptidase and epoxide hydrolase inhibitory activities were determined using recombinant
human LTA4H assays. The parent compound [6]-gingerol (N1) inhibits aminopeptidase activity
with an IC50 56.12 mM and inhibits epoxide hydrolase activity with an IC50 58.29 IC50. However,
4`-O-prenyl-[6]-gingerol (D10) showed the highest inhibition with an IC50 3.01 mM for
aminopeptidase and IC50 7.25 mM for epoxide hydrolase. Shogoal derivatives showed remarkable
LTA4H inhibition e.g. 4`-O-methyl shogoal (D8) showed aminopeptidase inhibitory activity with
an IC50 4.92 mM and epoxide hydrolase inhibitory activity with an IC50 11.27 mM. Other
gingerols that showed pronounced aminopeptidase inhibitory activity than N1 were [10]-gingerol
(N3),4`-O-acetyl shogoal (D2), 5,4`-diacetyl-[6]-gingerol (D3) and shogoal (D7) (Table 5).
Matching with the results of LTA4H inhibitory assays, compounds N3, D2, D3, D7 and
D10 showed remarkable cytotoxic activities with high selectivity index (SI) (SI; 3.00, 3.72, 3.23,
7.77 and 3.87, respectively) (Table 4 & 5).
10

In agreement to the docking results and the in vitro LTA4H inhibition assays, dimerization of [6]-
gingerol resulted in decrease in cytotoxic activity against HCT-116 cells in D9 (>100 mM).
Similarly, the breakage of the side chain of [6]-gingerol dramatically diminished the cytotoxic
activity as in methyl zingerone, D4 (>100 mM). Furthermore, the loss of aromaticity has a great
influence as expressed in 1,2-dihydroxybisabola-3,10-diene (N7) where the cytotoxic activity has
been dramatically diminished (>100 mM).
3. Materials and methods
3.1. General experimental information
Melting points were determined on Stuart® melting point apparatus model SMP10 and are
uncorrected. 1H and 13C-NMR spectra were obtained in CDCl3 solutions with TMS as internal
standardat 400 MHz for 1H-NMR and 100 MHz for 13C-NMR on BRUKER Avance III
spectrometer. Chemical shifts (d) are reported in ppm relative to the solvent signal and coupling
constants are given in Hz. Mass spectrometry, EI-MS spectra were obtained using a Thermo
Scientific DSQTM II instrument. While, ESI-MS experiments were conducted with 3200 Q-trap
LC/MS/MS system (Applied Biosystems) using Analyst version 1.4.1 software (MDS Sciex)
where analytes were ionized using electrospray ionization (ESI) interface operated in the
negative mode. IR spectra were obtained using a Thermo Scientific NicoletTM iSTM10 FT-IR
spectrometer instrument. The progress of reactions and the purity of final products were
monitored by thin layer chromatography (TLC) which is performed on precoated silica gel 60
GF254 (20 x 20 cm, 0.2 mm thick) on aluminium or plastic sheets (Merck, Germany), and
precoated reversed phase C18 silica gel glass plates, Partisil® KC18F Silica gel 60A with
fluorescent indicator (5 x 20 cm, 200 mm layer thickness). Whereas UV and vanillin/sulfuric acid
spray reagent were used as revealing agents. Column chromatography was carried out on silica
11

gel G 60-230 mesh (Merck, Germany) and reversed phase C18 silica gel (BAKERBOND®
octadecyl C18, 40 mm). Organic solvents were distilled prior use. All of the chemicals used in
these experiments were of reagent grade and obtained from commercial suppliers.
3.2. Plant material
The natural gingerols (N1-N5) used in this study was obtained from Z. officinaleby extraction
and isolation procedure described in our previous study.16
3.3. Semisynthesis of [6]-gingerol derivatives D1-D11
3.3.1. Preparation of compound D1 by Williamson ether synthesis.31
To a solution of [6]-gingerol (N1) (200 mg, 0.68 mmol, 1 equiv.) in acetone (5mL), allyl
bromide (82.3 mg, 1equiv.) and K2CO3 (187.7 mg, 1.36 mmol, 2 equiv.) were added and refluxed
at 50 ºC for 6 h The reaction was monitored by TLC [silica gel, petroleum ether-EtOAc (7:3)].
The reaction was stopped by addition of water (10 mL). The reaction mixture was extracted with
EtOAc (3x 10 mL). The EtOAc extract was dried over anhydrous sodium sulfate and evaporated
under vacuum at 45 ºC to obtain a crude residue.
3.3.2. Preparation of compounds D2 and D3.32, 33
To a solution of [6]-gingerol (N1) (200 mg) in methylene chloride (5mL), acetic anhydride (3
mL), and pyridine (0.1 mL) were added. The solution was stirred with a magnetic stirrer bar for 7
h at room temperature. The reaction was monitored by TLC [silica gel, petroleum ether-EtOAc
(8:2)]. The reaction was stopped by addition of water (10 mL). The reaction mixture was
extracted with methylene chloride (3x 10 mL). The methylene chloride extract was dried over
anhydrous sodium sulfate and evaporated under vacuum at 45 ºC to obtain a crude residue.
12

3.3.3. Preparation of compounds D4-D8.32
To a solution of [6]-gingerol (400 mg, 1.36 mmol, 1 equiv.) in methanol (15 mL), methyl iodide
(386 mg, 2 equiv.), and K2CO3 (375 mg, 2 equiv.) were added. The solution was stirred with a
magnetic stirrer bar for 48 h at room temperature. The reaction was monitored by TLC [silica
gel, petroleum ether-EtOAc (7:3)]. The reaction was stopped by addition of water (20 mL). The
reaction mixture was extracted with methylene chloride (3x 20 mL). The methylene chloride
extract was dried over anhydrous sodium sulfate and evaporated under vacuum at 45 ºC to obtain
a crude residue.
3.3.4. Preparation of compound D9.34
To a solution of [6]-gingerol (200 mg, 0.68 mmol) in methylene chloride (5 mL), 2.7 mL of
collins reagent were added with stirring in ice bath for 4 h Collins reagent was prepared by
dissolving CrO3 (2.5 g, 25 mmol, 1 equiv.) in dry pyridine (4 g, 50 mmol, 2 equiv.) with stirring
in ice bath, then addition of 10 mL of methylene chloride to the CrO3.2Py complex. The reaction
was monitored by TLC [C18 silica gel plates, methanol: water (7:3) system was used] . The
reaction was stopped by addition of EDTA solution (1gm EDTA in 10 mL water). The reaction
mixture was extracted with methylene chloride (3x 10 mL). The methylene chloride extract was
dried over anhydrous sodium sulfate and evaporated under vacuum at 45 ºC to obtain a crude
residue.
3.3.5. Preparation of compounds D10, and D11 by Williamson ether synthesis.31
To a solution of [6]-gingerol (100 mg, 1equiv.) in acetone (5 mL), (50.7 mg, 1 equiv.) of prenyl
bromide and (94 mg, 2 equiv.) of K2CO3 were added and refluxed at 50 ºC for 28 h The reaction
was monitored by TLC [silica gel, petroleum ether-EtOAc (2:1)] . The reaction was stopped by
13

addition of water (10 mL). The reaction mixture was extracted with methylene chloride (3x 10
mL). The methylene chloride extract was dried over anhydrous sodium sulfate and evaporated
under vacuum at 45 ºC to obtain a crude residue.
3.4. Chromatographic purification of [6]-gingerol derivatives D1-D11
3.4.1. Purification of allylation reaction product, D1
The crude residue obtained from reaction of allyl bromide with N1 was chromatographed over
silica gel (30 g) CC. using (petroleum ether-EtOAc, 9: 1 v/v, isocratic elution) to afford D1,
Rf0.5, petroleum ether-EtOAc, 7:3, v/v(187 mg, 93.4%).
3.4.2. Purification of acetylation reaction products, D2and D3
The crude residue obtained from acetylation reaction of N1 using acetic anhydride was
chromatographed over silica gel (25 g) CC. using (petroleum ether-EtOAc, 9.3: 0.7v/v, isocratic
elution) to afford D2, Rf0.5, petroleum ether-EtOAc, 8:2v/v (20 mg, 10%) and D3, Rf 0.35,
petroleum ether-EtOAc, 8:2v/v (80 mg, 40%).
3.4.3. Purification of methylation reaction products, D4-D8
The crude residue obtained from methylation reaction of N1 was chromatographed over silica
gel (25 g) CC. using (petroleum ether-EtOAc, gradient elution), and afforded two
fractions;fractions (1) and (2) eluted with petroleum ether-EtOAc (9.6: 0.4 v/v) and (9.5: 0.5
v/v), respectively. The selected fractions (1) and (2) were further subjected to MPLC using
reversed phase C18 silica gel. Fraction (1) eluted with (methanol: water, 7:3 v/v, isocratic) and
afforded D7, Rf 0.57, petroleum ether-EtOAc, 7:3 (5.5 mg, 1.4%) and D8, Rf 0.62, petroleum
ether-EtOAc, 7:3 v/v (15 mg, 3.7%). Fraction (2) eluted with (methanol: water, gradient elution)
afforded D4, Rf 0.33, petroleum ether-EtOAc, 7:3 v/v (18.6 mg, 4.6%) eluted with (methanol:
14

water, 6:4), while compounds D5, Rf 0.45, petroleum ether-EtOAc, 7:3 v/v (17.2 mg, 4.3%) and
D6, Rf 0.52, petroleum ether-EtOAc, 7:3 v/v (70.8 mg, 17.7%) were eluted with (methanol:
water, 7:3).
3.4.4. Purification of the oxidation reaction product, D9
The crude residue obtained from Collins oxidation of N1 was chromatographed over silica gel
(15 g) CC. using (petroleum ether-EtOAc, gradient elution) and afforded D9, Rf 0.34, petroleum
ether-EtOAc, 4:6 v/v (57.4 mg, 28.7%) eluted with (petroleum ether-EtOAc, 1: 1 v/v).
3.4.5. Purification of prenylation reaction products, D10 and D11
The crude residue obtained from reaction of prenyl bromide with N1 was chromatographed over
silica gel (15 g) CC. using (petroleum ether-EtOAc, gradient elution) to afford D11,Rf 0.75,
petroleum ether-EtOAc, 2:1 v/v (2 mg, 2%) eluted with (petroleum ether-EtOAc, 9.5: 0.5
v/v).and D10, Rf 0.42, petroleum ether-EtOAc, 2:1 v/v (91 mg, 91%) eluted with (petroleum
ether-EtOAc, 9: 1 v/v).
3.4.5.1. 4`-O-Allyl-[6]-gingerol (D1). Colorless needles with melting point 61-64 °C, IR (KBr)
13
nmax 3430, 2952, 2909, 2858, 1709, 1631,1516,1464, 1258, 1232, 1142, 1018cm-1;1H and C
●
NMR, see tables 1 and 2. EI-MSC20H30O4, m/z 334 [M]+, m/z 316 [M- H2O]+ , m/z 275, 234,
193, 177, 149, 137, 119, 107, 91, 77.
1
13
3.4.5.2. 5-O-Methyl-[6]-gingerol (D5). Pale yellow oily liquid, H and C NMR, see tables 1
and 2. EI-MSC18H28O4, m/z 308 [M]+, m/z 276 [M- CH3OH]+●, m/z 205, 179, 150, 137, 115, 91.
3.4.5.3. Dimethyl-[6]-gingerol (D6). Pale yellow oily liquid, IR (KBr) nmax 2931, 2860, 2834,
1712, 1608, 1590, 1515, 1463, 1417, 1366, 1262, 1189, 1153, 1093, 1030, 937, 852, 806, 765
15

1
cm-1; H and 13C NMR, see tables 1 and 2. EI-MSC19H30O4, m/z 322 [M]+, m/z 290 [M-
CH3OH]+●, m/z 259, 219, 193, 164, 151, 121, 115.
→II
3.4.5.4. Bis-(I-5`
-5`)-[6]-gingerol (D9). The obtained product was a pale yellow amorphous
powder with melting point 96-98 °C,IR (KBr) nmax 3434, 2957, 2927, 2855, 1708, 1601, 1450,
1
13
1379, 1254, 1147, 1049, 919 cm-1; H and C-NMR, see tables 1 and 2. LC/MS/MS[M-H]+ at
m/z 585.4(calculated for C34H50O8, 586.35).
3.4.5.5. Prenyl-[6]-gingerol (D10). White powder with melting point 48-50 °C, IR (KBr) nmax
3429, 2989, 2923, 2855, 1707, 1629, 1606, 1513, 1468, 1380, 1225, 1136, 1085, 1075, 1034, 993
cm-1; 1H and 13C NMR, see tables 1 and 2. EI-MSC22H34O4, m/z 362 [M]+, m/z 344 [M- H2O]+●,
m/z 294, 194, 179, 150, 137, 119.
3.5. Docking study:
3.5.1. Ligand and protein preparation
The structures of [6]-gingerol and its derivatives were constructed using ChemBio3D Ultra 12.0
and saved as SDF files. While, bestatin SDF file was downloaded from Pubchem database
(https://pubchem.ncbi.nlm.nihr.gov). Their energy forms were minimized using uff force field,
and converted to pdbqt format by Open Babel in PyRx 0.8 to be used as ligands for virtual
screening. On the other side, the protein structure was prepared using PyRx 0.8.Additionally,
4BS and other non-protein molecules were removed except for zinc atomas it participates in the
interaction between the receptor and the ligand.
3.5.2. Virtual screening
With the aim to investigate different theoretical binding modes of our ligands (gingerol
derivatives), molecular modeling study was performed by means of AutoDock Vina in PyRx 0.8
using LTA4H enzyme which was retrieved from RCSB-Protein Data Bank (PDB code: 3CHR).
16

The docking site for the ligands on LTA4H was defined by establishing a cube at the geometrical
center of the native ligand present in the evaluated PDB structure with the dimensions 25x 25 x
25 Å, covering the ligand binding site. The coordinates X, Y and Z from center grid box were
9.0929, 39.8746 and 67.9694, respectively.
3.5.3. Visualization
All the visualization of the docking files were done using PyMOL molecular graphics system
(www.pymol.org).
3.6. Cytotoxicity assay 35,36
3.6.1. Materials
All cell lines; human colon cancer cells (HCT-116), normal human fibroblast cells (TIG-1) and
normal human fetal lung fibroblast cells (HF-19) were obtained from Riken Bioresource Center
of Japan (Ibaraki, Japan). HCT-116 cells were cultured in McCoy’s 5A Medium (Wako Pure
Chemical Industries, Osaka, Japan), TIG-1 and HF-19 were cultured in Dulbecco’s Modified
Eagle’s medium (DMEM) (Wako Pure Chemical Industries, Osaka, Japan) supplemented
with 10% fetal bovine serum, 100 U/ml penicillin, and 100 U/ml streptomycin (Gibco BRL,
Tokyo, Japan).
3.6.2. Cell culture
HCT-116, HF-19 and TIG-1 cells were cultured in 96-well plates (Greiner Bio-One) at density of
2 × 103 cells/well, respectively, in a humid atmosphere of 5% (v/v) CO2 and 95% (v/v) air at
37°C. After 48h, the cells were treated with five different concentrations of compounds (6.25,
12.5, 25, 50, and 100 μM, dissolved in DMSO). Bestatin and 4-(4-Benzylphenyl)-thiazol-2-
amine (4BSA) (Merck Millipore, Germany) were used as a positive controls. After 48 h, cell
17

viability was determined using WST-1 reagent as follows: 10 μL of WST-1 reagent was added to
each well, followed by 4 h incubation at 37°C. All incubations were carried out in triplicate, the
absorbance was measured at 450 nm using a Microplate Reader (Biotek, Winooski, VT, USA).
3.7. Aminopeptidase assay 37
Aminopeptidase activity was determined by modification of Rao et al. (2007).37 Recombinant
human LTA4H (Cayman, Cat. no. 10009292, Ann Arbor, MI, USA) (415 ng) was pre-incubated
for 20 min at room temperature in the assay buffer (50 mM tris HCL, 100 mM KCl , PH= 8) in
the presence of different concentrations of the tested compounds (0.2% final DMSO
concentration). The substrate; L-alanine-4-nitro-anilide hydrochloride; A9325 was added to the
reaction mixture at a final concentration 1.25 mM. Aminopeptidase activity was determined by
monitoring the absorbance at 405 nm. IC50 values were calculated by nonlinear regression using
GraphPad Prism 6.0(GraphPad Software, Inc. San Diego, CA).
3.8. Epoxide hydrolase Activity:
Recombinant human LTA4H (50 ng) was incubated with various concentrations of test
compounds (dissolved in DMSO, final DMSO concentration is 0.1% ) for 20 min at room
temperature in assay buffer (0.1 M potassium phosphate, pH 7.4, 5 mg/ml fatty acid-free bovine
serum albumin) in a volume of 50 µL. The solution was then adjusted to 200 µL with assay
buffer, and 50 µL of the substrate, LTA4, was added (final concentration, 50 ng/ml, final
volume, 250 µL). After 30 min at room temperature, the assay was terminated by diluting 20-
fold in assay buffer. The amount of LTB4 produced was assayed by enzyme immunoassay (EIA)
(Cayman, Cat. no. 10009292, Ann Arbor, MI, USA) according to the manufacturer’s protocol.
IC50 (the concentration of compounds required for the half maximal inhibition of the enzyme
18

activity determined by the amount of LTB4 production against negative control) values were
calculated as described above for aminopeptidase assay.
4. Conclusion
Eleven semisynthesized [6]-gingerol derivatives along with seven naturally isolated derivatives
including [4]-, [6]-, [8]-, and [10]-gingerol were docked into the catalytic site of LTA4H protein.
Also, their cytotoxicity against colon cancer cells (HCT-116) and normal cells (TIG-1 & HF-19)
were investigated. The active compounds showed selective cytotoxicity against cancer cells.
Their aminopeptidase and epoxide hydrolase inhibitory activity of LTA4H were evaluated.
Shogaol derivatives; 4`-O-acetyl shogoal (D2), shogoal (D7) and 4`-O-methyl shogoal (D8)
showed better inhibitory activity compared to other gingerol series. Nevertheless, [10]-gingerol,
diacetyl-[6]-gingerol (D3) and 4`-O-prenyl-[6]-gingerol (D10) showed better activities than that
[6]-gingerol, N1. These results are in full agreement with docking and in vitro cytotoxicity
studies and presented a lead for new selective natural aminopeptidase and epoxide hydrolase
inhibitors as potential therapeutic agents for colorectal cancer.
Supplementary data
13C- and1H-NMR of N1, D1, D3, D5, D6, D9, D10, D11, N6, 2D-NMR of N6 and LC/MS/MS
spectra of compound D9.
19

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22

Figures captions
Figure 1. (A) The final rate-limiting step in the biosynthesis of leukotriene B4 and (B) Model of
LTA4H interactions as epoxide hydrolase3.
Figure 2.Chemical structures of natural and semisynthetic gingerol derivatives.
Figure3. (A) Overlap of [6]-gingerol (red), bestatin (blue) and 4BS (yellow) within LTA4H
catalytic site; (B) Detailed view of docked structure N1 and(C) of N6 with the corresponding
interacting amino acids and the catalytic zinc ion (gray sphere) of LTA4H; (D) Overlay of N1
(red), N2 (blue), and N3 (yellow) within LTA4H catalytic site.
Figure 4. Detailed view of docked structure D5 (A), D7 (B), D1 (C) with the corresponding
interacting amino acids and the catalytic zinc ion (gray sphere) of LTA4H; (D) Molecular model
of D9 (yellow), in comparison to that of N1 (red) within LTA4H catalytic site.
23

Table 1. 1H-NMR spectra for [6]-gingerol derivatives; D1, D3, D5, D6, D9, D10, and D11 in
comparison to [6]-gingerol (N1)*
dH
H#
1
D9 ♦
N1
D5
D6
D3
D1
D10
D11
2.88 (4H,
overlapped
m)
2.79 (2H,br d,
J=6.8 Hz)
2.88 (2H, t,
J=7.0Hz)
2.74 (2H, brd,
J=7.1 Hz)
2.76 (2H, br d,
2.78 (2H, t,
J= 7.7 Hz)
2.76 (2H, t,
J=7.2 Hz)
2.85 (2H, t,
J=7.2 Hz)
J=7.1 Hz)
2.88 (4H,
overlapped
m)
2.71 (2H, br d,
J=6.8 Hz)
2.77 (2H, t,
J=7.0 Hz)
2.68 (2H, br d,
J=6.8 Hz)
2.67 (2H, t,
J= 7.7 Hz)
2.66 (2H, t,
J=7.2 Hz)
2.74 (2H, t,
J=7.2 Hz)
2
3
2.65 (2H, m)
---
---
---
---
---
---
---
---
4a: 2.48 (1H,
4a: 2.31 (1H,
dd, J=4.8, 15.7
Hz)
4b: 2.54 (1H,
dd, J=7.6, 15.7
Hz)
4a: 2.31 (1H,
4a: 2.46 (1H,
4a: 2.47 (1H,
d, J=8.9, 17.5
Hz)
4b: 2.57 (1H,
d, J=2.9, 17.4
Hz)
4a: 2.43 (1H, dd, dd, J=8.8,
J=8.7,17.4 Hz) 17,4 Hz)
4b: 2.52 (1H, dd, 4b: 2.58 (1H,
dd, J= 4.5, 15.7 dd, J=5.2, 16 4a: 2.38 (1H, dd,
Hz)
Hz)
4b: 2.61 (1H, 4b: 2.48 (1H, dd,
dd, J= 7.5, 15.7 dd, J=7.4, J=2.4, 17.6 Hz)
J=8.8, 17.6 Hz)
6.10 (1H, d,
J=15.1 Hz)
4
4b: 2.54 (1H,
J=3.2,17.8 Hz)
dd, J=2.8,
17.3 Hz)
Hz)
16.6 Hz)
5
6
3.99 (1H, m)
4.00 (1H, m)
3.56 (1H, m)
3.56 (1H, m)
1.30-1.41 (2H,
m)
5.13 (1H, m) 3.94 (1H, m)
1.35 (2H, d,
J=5.2 Hz )
4.04 (1H, m)
6.85 (1H, m)
2.19 (2H, q,
J=8.2 Hz)
1.44 (2H, m)
1.24-1.33
1.30 (2H, m)
7
8
9
1.25-1.30 (8H,
m)
1.30-1.50
(8H, m)
1.22-1.40 (8H,
1.27-1.51
(8H, m)
1.20-1.25 (6H,
m)
1.20-1.30 (6H,
m)
1.18-1.34
m)
(6H, m)
(4H, m)
0.86 (3H, t,
J=6.88 Hz)
0.88 (3H, t,
J=6.7 Hz)
0.80 (3H, t, J=7 0.79 (3H, t,
0.79 (3H, t,
J=6.4 Hz)
---
1.92 (3H, s)
---
0.80 (3H, t,
J=6.8 Hz)
0.89 (3H, t,
J=6.8 Hz)
0.90 (3H, t,
J=6.9 Hz)
10
Hz)
J=6.7 Hz)
11
12
1'
3.22(3H, s)
3.21(3H, s)
---
---
---
---
---
---
---
6.65 (1H,d,
J=1.8Hz)
---
6.73 (2H,br
s)
---
---
6.62 (1H, d,
J=1.7 Hz)
---
6.65 (2H, brd
s)
---
---
2'
6.72 (1H,s)
6.63 (1H,s)
6.71 (1H, s)
6.75 (1H,s)
3'
4'
---
---
---
---
---
---
---
---
---
---
6.80 (1H, d,
J=7.8Hz)
6.74 (1H, d,
J=7.9Hz)
6.70 (1H, d,
J=8.6 Hz)
6.84 (1H, d,
J=8 Hz)
6.71 (1H, d,
J=8.4 Hz)
6.80 (1H, d,
J=8 Hz)
6.81 (1H, d,
J=7.2 Hz)
5'
---
6.68 (1H,
overlapped d,
J=8.5 Hz)
3.86 (3H, s)
4.55 (2H, d,
J=6.6 Hz)
5.51 (1H, t,
J=6.5 Hz )
6.62 (1H, dd,
J=7.8,1.8 Hz)
6.73 (2H, br
s)
6.58 (1H, dd,
J=7.9,1.7 Hz)
6.65 (2H, brd
s)
6.66 (1H, d,
J=8 Hz)
6.59 (1H, d, J=8
Hz)
6.72 (1H, d,
J=8.8 Hz)
6'
7'
8'
3.84(3H, s)
3.92 (3H, s)
3.79(3H, s)
3.77 (3H, s)
3.79 (3H, s)
3.74 (3H, s)
---
3.79 (3H, s)
4.50 (2H, d,
J=5.6 Hz)
3.87 (3H, s)
4.56 (2H, d,
J=6.3 Hz)
5.52 (1H, t,
J=6 Hz )
9'
2.23 (3H, s)
5.96 (1H, m)
10`a: 5.19 (2H,
d, J=10.4Hz)
10`b: 5.30 (2H,
d, J=17.6Hz)
10`
---
---
11`
12`
1.74 (3H, s)
1.78 (3H, s)
1.74 (3H, s)
1.79 (3H, s)
* In CDCl3, at 400 MHz, Coupling constants (J) are in Hz.
♦The values are corresponding to equivalent positions in the two units of the dimer.
24

Table 2. 13C-NMR spectra for [6]-gingerol derivatives; D1, D3, D5, D6, D9, D10, and D11 in
comparison to [6]-gingerol (N1)*
dc
D6
C#
N1
29.4, CH2
45.5, CH2
211.6, qC
49.4, CH2
67.7, CH
36.5, CH2
25.2, CH2
31.8, CH2
22.7, CH2
14.1, CH3
D9
D5
D3
29.4
44.8
206.8
47.3
70.3
34.2
24.8
31.5
22.5
D1
28.2
44.3
210.4
48.3
66.6
35.4
24.1
30.7
21.6
13.0
D10
D11
1
2
3
4
5
6
7
8
29.3, CH2
45.3, CH2
211.4, qC
49.4, CH2
67.7, CH
36.5, CH2
25.1, CH2
31.7, CH2
22.6, CH2
14.0, CH3
29.3, CH2
45.8, CH2
209.0, qC
47.6, CH2
77.3, CH
33.8, CH2
24.7, CH2
31.9, CH2
22.6, CH2
14.0, CH3
56.9, CH3
29.2, CH2
45.7, CH2
208.9, qC
47.6, CH2
77.3, CH
33.8, CH2
24.7, CH2
31.9, CH2
22.6, CH2
14.0, CH3
56.9, CH3
29.2, CH2
45.3, CH2
211.4, qC
49.4, CH2
67.7, CH
36.5, CH2
25.1, CH2
31.7, CH2
22.6, CH2
14.0, CH3
29.8, CH2
41.9, CH2
199.8, qC
130.3, CH2
147.8, CH
32.4, CH2
27.8, CH2
31.3, CH2
22.4, CH2
13.9, CH3
9
10
11
12
1'
2'
3'
4'
5'
6'
7'
8'
9'
10`
11`
12`
14.0
170.5
21.1
132.7, qC
111.0, CH
146.4, qC
144.0, qC
114.4,CH
120.8, CH
55.9, CH3
132.6, qC
110.6, CH
141.0, qC
147.3, qC
124.4, qC
122.7, CH
56.1, CH3
133.0, qC
111.1, CH
146.4, qC
144.0, qC
114.3,CH
120.8, CH
55.9, CH3
133.8, qC
111.8, CH
148.9, qC
147.4, qC
111.3,CH
120.1, CH
55.8, CH3
55.9, CH3
140.0
112.7
150.9
138.0
122.6
120.4
55.8
132.5
112.6
148.4
145.4
111.0
119.0
54.9
68.9
132.8
116.9
133.4, qC
113.4, CH
149.5, qC
146.7, qC
111.9,CH
120.1, CH
55.9, CH3
65.9, CH2
120.0, CH
137.4, CH2
18.2, CH3
25.8, CH3
134.1, qC
113.3, CH
149.4, qC
146.6, qC
112.1,CH
120.2, CH
55.9, CH3
65.9, CH2
120.0, CH
137.4, CH2
18.2, CH3
25.8, CH3
169.2
20.7
* In CDCl3, at 100 MHz. Carbon multiplicities were determined by APT experiment. qC =
quaternary, CH = methine, CH2 = methylene, CH3 = methyl carbons.
♦The values are corresponding to equivalent positions in the two units of the dimer.
25

Table 3. Docking scores of gingerol derivatives towards the active site of LTA4H (PDB code:
3CHR), using AutoDock Vina in PyRx 0.8.
Compound
Binding energy(Kcal/mol)a Compound Binding energy (Kcal/mol)
-8.8
-8.9
-9.3
-8.9
-8.9
-9.4
-7.4
-8.6
-9.0
-8.6
-7.5
-8.7
-8.6
-9.1
-8.8
-8.8
-8.9
-8.6
-9.2
-10.1
-11.4
N1 (Gingerol)
D5
D6
N2
N3
N4
N5
N6
N7*
D1
D2
D3
D4
D7
D8
D9
D10
D11
Bestatin
4BS
4BSA
aA more negative score indicates that a molecule is more likely to dock with the structure and
achieve more favorable interactions.29
*The stereochemistry for compound N7 couldn’t be determined. However, it was proposed as shown in
figure 2 to enable its docking study.
26

Table 4: Cytotoxicity of compounds against HCT-116 and two normal cell lines; TIG-1 and HF-
19. All data are presented as mean value ± SD for three independent experiments.
In vitro Cytotoxicity IC50 (µM)
Compounds
HCT-116
42.54 ± 1.58
30.54 ± 0.78
45.05 ± 2.57
> 100
TIG-1
77.82 ± 1.38
84.28 ± 2.21
75.17 ± 4.12
55.64 ± 4.22
> 100
HF-19
89.51 ± 5.04
> 100
SI**
1.83
2.76
1.67
0.55
3.00
1.00
1.21
2.17
1
Bestatin*
4BSA*
N1
> 100
77.62 ± 2.51
> 100
N2
33.36 ± 0.27
> 100
N3
> 100
> 100
N4
92.78 ± 4.28
> 100
88.36± 1.18
97.54 ± 2.37
> 100
76.49 ± 3.15
46.03± 1.23
> 100
N5
N6
> 100
N7
64.58 ± 1.89
> 100
> 100
0.65
3.72
3.23
1.00
1.00
1.00
7.77
52.3
0.63
3.87
> 100
D1
26.87 ± 2.75
30.95 ± 1.54
> 100
> 100
D2
> 100
> 100
D3
> 100
> 100
D4
> 100
> 100
D5
> 100
> 100
> 100
> 100
D6
> 100
> 100
12.87 ± 0.41
1.54 ± 1.24
> 100
D7
80.57 ± 6.24
63.12 ± 5.15
83.24 ± 3.51
> 100
D8
54.8 ± 3.15
> 100
D9
21.51 ± 1.02
D10
* Bestatin and 4BSA were used as positive controls.
**SI refers to Selectivity Index, calculated compared to the normal TIG-1 cells, SI value > 3
indicating high selectivity.
27

Table 5: The inhibition of LTA4H aminopeptidase activity and LTB4 production by the tested
compounds. All data are presented as mean value ± SD for three independent experiments.
Aminopeptidase
inhibition
Epoxide hydrolase
inhibition
Compounds
(IC50 µM)
18.01 ± 0.26
12.4 ± 0.12
58.29 ± 0.48
> 100
(IC50µM)
10.10 ± 0.01
7.61 ± 0.4
56.12 ±0.22
> 100
Bestatin*
4BSA*
N1
N2
21.59 ± 1.02
> 100
15.24 ± 0.84
> 100
N3
N4
> 100
> 100
N5
45.61 ± 0.84
> 100
67.12 ±1.20
> 100
N6
N7
56.27 ± 1.28
9.31 ± 0.12
22.11 ± 0.82
> 100
66.43 ± 1.51
29.47 ± 0.43
27.62 ± 0.79
> 100
D1
D2
D3
D4
> 100
> 100
D5
> 100
> 100
D6
12.54 ± 0.18
4.92 ± 0.72
> 100
24.61 ± 0.45
11.27 ± 0.51
77.82 ± 2.47
7.25 ± 0.13
D7
D8
D9
3.01 ± 0.014
D10
* Bestatin and 4BSA were used as positive controls
28

H
O
O
O
H
(A)
O
H
Leuckotriene A4
CH3
O
H
H
O
O
H
O
CH3
Leuckotriene B4
(B)