Design, synthesis, and anticancer evaluation of long-chain alkoxylated mono-carbonyl analogues of curcumin

Article abstract of DOI:10.1016/j.ejmech.2015.08.036

Curcumin is a nontoxic phenolic compound that modulates the activity of several cellular targets that have been linked with cancers and other chronic diseases. However, the efficacy of curcumin in the clinic has been limited by its poor bioavailability an

Full text of DOI:10.1016/j.ejmech.2015.08.036

Accepted Manuscript
Design, Synthesis, and Anticancer Evaluation of Long-chain Alkoxylated Mono-
carbonyl Analogues of Curcumin
Qiaoyou Weng, Lili Fu, Gaozhi Chen, Junguo Hui, Jingjing Song, Jianpeng Feng,
Dengjian Shi, Jiansong Ji, Professor, Guang Liang, Ph.D, Professor Director
PII:
S0223-5234(15)30220-8
10.1016/j.ejmech.2015.08.036
EJMECH 8072
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 April 2015
Revised Date: 12 August 2015
Accepted Date: 15 August 2015
Please cite this article as: Q. Weng, L. Fu, G. Chen, J. Hui, J. Song, J. Feng, D. Shi, J. Ji, G. Liang,
Design, Synthesis, and Anticancer Evaluation of Long-chain Alkoxylated Mono-carbonyl Analogues of
Curcumin, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.08.036.
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ACCEPTED MANUSCRIPT
Design, Synthesis, and Anticancer Evaluation of Long-chain
Alkoxylated Mono-carbonyl Analogues of Curcumin
Qiaoyou Weng1,2#, Lili Fu1#, Gaozhi Chen1, Junguo Hui2, Jingjing Song2, Jianpeng Feng1, Dengjian Shi1, Jiansong
Ji2*, Guang Liang1*
1. Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University,
Wenzhou, Zhejiang 325035, China
2. Department of Interventional Radiology, The Fifth Affiliated Hospital of Wenzhou Medical University,
Lishui, Zhejiang 323000, China
#These Authors contributed equally to this work
* Corresponding authors:
Guang Liang, Ph.D, Professor
Director, Chemical Biology Research Center
School of Pharmaceutical Sciences, Wenzhou Medical University
Tel: (+86)-577-86699396; Fax: (+86)-577-86699396
E-mail: wzmcliangguang@163.com
and
Jiansong Ji, Professor
Department of Interventional Radiology, the Fifth Affiliated Hospital of Wenzhou Medical University
E-mail: jjstcty@sina.com

ACCEPTED MANUSCRIPT
Graphical abstract
Novel curcumin analogues were synthesized and revealed as potent anti-cancer candidate.
Keywords: anticancer drug, gastric cancer, drug design, MACs, apoptosis

ACCEPTED MANUSCRIPT
Design, Synthesis, and Anticancer Evaluation of Long-chain
Alkoxylated Mono-carbonyl Analogues of Curcumin
Qiaoyou Weng1,2#, Lili Fu1#, Gaozhi Chen1, Junguo Hui2, Jingjing Song2, Jianpeng Feng1, Dengjian Shi1, Yuepiao
Cai1, Jiansong Ji2*, Guang Liang1*
1. Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University,
Wenzhou, Zhejiang 325035, China
2. Department of Interventional Radiology, The Fifth Affiliated Hospital of Wenzhou Medical University,
Lishui, Zhejiang 323000, China
#These Authors contributed equally to this work
* Corresponding authors:
Guang Liang, Ph.D, Professor
Director, Chemical Biology Research Center
School of Pharmaceutical Sciences, Wenzhou Medical University
Tel: (+86)-577-86699396; Fax: (+86)-577-86699396
E-mail: wzmcliangguang@163.com
and
Jiansong Ji, Professor
Department of Interventional Radiology, the Fifth Affiliated Hospital of Wenzhou Medical University
E-mail: jjstcty@sina.com
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Abstract: Curcumin is a nontoxic phenolic compound that modulates the activity of several cellular targets that
have been linked with cancers and other chronic diseases. However, the efficacy of curcumin in the clinic has been
limited by its poor bioavailability and rapid metabolism in vivo. We have previously reported the design and
discovery of series of 5-carbon linker-containing mono-carbonyl analogues of curcumin (MACs) as anti-cancer
agents. In continuation of our ongoing research, we designed and synthesized 37 novel long-chain alkoxylated
MACs for anti-cancer evaluation here. The MTS assay was used to determine the cytotoxicity of compounds in
gastrointestinal cancer cells. Compounds 5, 28, and 29 showed strongest inhibition against gastric cancer cell
proliferation and were subjected to further analysis. The effects of 5, 28, and 29 on cell apoptosis were measured
by flow cytometry. Expression levels of Bcl-2, cleaved poly ADP-ribose polymerase (PARP), and pro-caspase-3
were detected by western blotting. Compounds 5, 28, and 29 induced apoptosis in human gastric carcinoma cells,
increased PARP cleavage, and decreased expression of Bcl-2 and cleavage of PARP protein. We then showed that
compound 28, which possessed the strongest activity among the test compounds in vitro, exhibited significant
tumour inhibition in an SGC-7901-driven xenograft mouse model. Taken together, the novel compound 28 could be
further explored as an effective anticancer agent for the treatment of human gastric cancer.
Keywords: anticancer drug, gastric cancer, drug design, MACs, apoptosis.
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1. Introduction
Despite a sharp decline in its incidence during the second half of the 20th century, gastric cancer remains the
fourth most frequently diagnosed type of cancer and the second most common cause of cancer-related deaths
worldwide[1-3] ,with 988,000 new cases and 736,000 deaths per year[4-6]. Almost two-thirds of gastric cancer cases
occur in developing countries, and 42% of cases occur in China[7]. Prognosis of gastric cancer is generally poor with
a 5-year relative survival below 30% in most countries[8]. Surgery and chemotherapy are the most popular therapy
for gastric cancer [9, 10]. Surgical resection is the primary treatment for gastric cancer and it can cure the patients
with early-stage cancer. Yung-Jue Bang and colleagues reported a 5-year survival rate of 36% in patients with
operable disease who received perioperative chemotherapy. However, the 5-year survival rate for advanced or
metastatic gastric cancer patients is around 5–20%[11]. Gastric cancer at an early stage may be clinically silent, and
in most countries, patients are diagnosed at an advanced stage when the tumor is unresectable or metastatic. For
these patients, as well as disease recurrence after initial surgical treatment, systemic chemotherapy is the main
treatment option even though it prolongs survival without compromising quality of life[12].
Although a large number of chemotherapy regimens have been tested in randomized clinical studies, there is
still no internationally accepted standard for the gastric cancer treatment. Existing chemotherapeutic formulations
containing fluoropyrimidine and cisplatin do not have ideal curative effects and have many undesirable side effects
[13-15]. Therefore, most gastric cancer patients who diagnosed with inoperable advanced or metastatic cancer
require palliative care. Development of pharmacologically effective agents with acceptable toxicity profiles has
become an urgent demand.
Numerous types of anti-cancer compounds exist in edible and medicinal plants, and chemical and
pharmacological investigations of such compounds are in progress [16-18]. The phenolic diarylheptanoid
curcumin is the major pigment in turmeric [19] and has been found to inhibit multiple cancer types via its effect on
several biological pathways involved in mutagenesis, cell cycle regulation, oncogene expression, apoptosis,
tumorigenesis, and metastasis [20]. These findings regarding the molecular mechanisms of curcumin shows that it
worth to further consideration as a treatment for cancer. However, rapid plasma clearance and low bioavailability
significantly limit the therapeutic application of curcumin [21]. Due to the metabolic instability, the β-diketone
moiety of curcumin was displaced with a single carbonyl group in our previous study, and a series of
mono-carbonyl analogues of curcumin (MCACs) were synthesized and evaluated the pharmacokinetics and
cytotoxicity. Some MCACs exhibited enhanced stability in vitro and greatly improved pharmacokinetic profiles in
vivo. Some MCACs exhibited higher cytotoxic than the lead compound curcumin. The structure-activity
relationship analysis in our previous work showed that MCACs with electron-withdrawing substitutions or weak
electron-donating substitutions increased cytotoxicity (Figure 1)[22]. Furthermore, due to the frequently presence
of a long-chain alkoxyl group in several anticancer drugs, such as foretinib, gefitinib, cediranib, and SKI-606, we
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proposed that the MCACs with long-chain alkoxyl groups might have more increased pharmacological activity in
comparison with other MCACs. Thus, in the present study, we designed a series of long-chain alkoxylated
mono-carbonyl analogues of curcumin and tested their anti-tumour activity in vivo and in vitro.
Please insert Figure 1
2. Results and Discussion
2.1 Chemistry
The common synthetic route and structures of compounds 1–22 are outlined in Figure 2. Intermediate 2 was
prepared from hydroxybenzaldehyde and aliphatic dibromoalkanes in 1 step. Intermediate 2 was refluxed with
appropriate amines in basic conditions to afford intermediate 3. Compounds 1–22 were prepared by the aldol
condensation reaction of cyclic ketones and intermediate 3 with yields of 15.4%–72.9%. For the synthesis of
compounds 23–37 (Figure 3), intermediate 4 was first produced through the aldol condensation reaction of
intermediate 3 and acetone in a basic solution. A one-step aldol condensation reaction was used to produce
compounds 23–37 (24.3%–76.8%) using substituted benzaldehydes and intermediate 4.
The structures of all compounds were characterized using 1H-NMR and electrospray ionization mass
spectrometry (ESI-MS) and three most active compounds were further characterized by 13C-NMR. Before they were
used in biological experiments, all synthesized compounds were purified by recrystallization or silica gel column
chromatography, and HPLC was used to determine their purity (all >95%).
Please insert Figure 2
Please insert Figure 3
2.2 Inhibitory Activity on Gastrointestinal Carcinoma Cells of All Compounds
The anti-proliferative effects of compounds 1−37 in 4 gastrointestinal carcinoma cell lines (MGC803,
SGC7901, CT26, and SW620) were determined by MTS assay (Promega, San Luis Obispo, CA, USA) at a
concentration of 10 ꢀM (Table S1). Surprisingly, 20 of the test compounds inhibited the growth of the 4
gastrointestinal carcinoma cell lines by more than 50% at a concentration of 10 ꢀM, and the IC50s of these 20
compounds against the MGC803, SGC7901, CT26, and SW620 cell lines were determined (Table 1). The 20 tested
compounds showed excellent anti-proliferative activity in 4 gastrointestinal carcinoma cell lines (IC50 = 0.18−8.46
ꢀM), with the exceptions of compounds 2 and 9. One of the most noteworthy results of this experiment was that
compounds 5, 16, 18, 28, and 29 inhibited SGC-7901 cells more effectively than the other tested compounds. Further
evaluation of the toxicity and physicochemical properties of compounds 5, 16, 18, 28, and 29 were shown in Figure
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S1-S5. Compounds16 and 18 showed poor solubility at 3.63µg/ml and 4.11µg/ml, much lower than that of
compounds 5, 28, and 29 (21.65µg/ml, 21.23µg/ml, and 113.5µg/ml, respectively). Thus, in consideration of their
toxicity, solubility, and activity, compounds 5, 28, and 29 were chosen for further studies.
Please insert Table 1
2.3 Induction of Apoptosis by compounds in Vitro
The effects of compounds 5, 28, and 29 on SGC-7901 tumour cell apoptosis were tested, curcumin served as a
positive control. SGC-7901 tumour cells were treated with various concentrations of the test compounds or vehicle
for 30 h. The cells were harvested and stained with Annexin-V and PI, the proportions of apoptotic cells in each
group were determined by flow cytometry analysis. Compounds 5 and 28 significantly and dose-dependently
increased apoptosis in SGC7901 cells in comparison with the negative control cells (Figure 4), while compound 29
did not exhibit this activity. By comparing the pro-apoptotic effects of the test compounds, we found that compound
28 exhibited a superior pro-apoptotic activity and that its effect was dose-dependent. Compound 28 induced
apoptosis in approximately 50% of treated SGC7901 cells at a concentration of 10 ꢀM, demonstrating that it was the
most effective of the tested compounds in SGC7901 cells.
Please insert Figure 4
2.4 Effects of compounds on apoptosis proteins
To determine the mechanisms underlying the pro-apoptotic activity of the test compounds, we examined the
effects of compounds 5, 28 and 29 on apoptosis-related proteins in SGC7901 cells, using curcumin as a control
treatment. SGC7901 cells were treated with various concentrations of compounds 5, 28, and 29. Expression and
activation of apoptosis-related signalling molecules (cleaved PARP, Bcl-2, and pro-caspase-3) were determined by
immunoblotting (Figure 5). The caspase-3 signalling pathway, a pro-oncogenic pathway, plays a key role in
regulation of cell functions via regulation of genes involved in cell proliferation [23]. Activation of caspase-3 was
indicated by a decrease in the abundance of pro-caspase-3 fragments. As shown in Figure 5, caspase-3 activation
was observed after exposure to compounds 5, 28, and 29. Bcl-2 family proteins, which have either pro- or
anti-apoptotic activities, have been studied intensively for the past decade because of their importance in the
regulation of apoptosis, tumorigenesis, and cellular responses to anti-cancer therapy[24]. In the Bcl-2 protein family,
anti-apoptotic member Bcl-2 is the active effector and regulator which affect apoptosis induction [25, 26]. Treatment
with compounds 5 and 28 significantly decreased Bcl-2 expression. The presence of cleaved PARP is generally
considered a marker of apoptosis [27]. Compounds 5 and 28 increased cleaved PARP abundance in SGC7901 cells
in a dose-dependent manner, suggesting increased apoptosis 24 hours after treatment with the compounds. In
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accordance with the flow cytometry analysis, compound 28 was more potent than compound 5, while compound 29
produced no significant change in cleaved PARP at any concentration, suggesting that compound 28 possessed
optimal growth inhibitory and pro-apoptotic activities in the SGC7901 cells.
Please insert Figure 5
2.5 In Vivo Antitumor Activity
In consideration of the results from the apoptosis and immunoblotting assays, compound 28 was selected for
further evaluation of in vivo antitumor efficacy in an SGC-7901 human gastric carcinoma cell xenograft mouse
model (Figure 6). Nude mice bearing established SGC-7901 tumour xenografts were dosed orally with compound
28 (50 mg/kg or 100 mg/kg daily over a 23-day period). Curcumin (50 mg/kg) was used as a positive control drug.
Relative to the vehicle-treated control group, the 100 mg/kg/day dose of compound 28 showed significant in vivo
antitumor efficacy (48%) and produced no clinical signs of intolerability. Moreover, tumour weight was
dose-dependently decreased by compound 28 (Figure 6B). During the experiment, no significant loss of body
weight was observed in mice treated with compound 28 in comparison with the vehicle control group (Figure 6C).
Immunohistochemical analysis performed on tumour sections confirmed that treatment with compound 28
significantly decreased cell proliferation (Ki67 staining) and increased apoptosis (cleaved-caspase-3) in the
tumour-bearing mice (Figure 7).
Please insert Figure 6
Please insert Figure 7
3. Conclusion
In this study, we reported the synthesis of a series of long-chain alkoxylated MCACs and evaluation of their
biological activity. All of the compounds showed inhibitory effects towards gastrointestinal carcinoma cells, which
were superior to that of curcumin. Compounds 5, 28, and 29 showed strong inhibition of SGC-7901 cells, which was
superior to that produced by curcumin. The anti-proliferative activity of the test compounds in gastrointestinal
carcinoma cells was tested using the MTS assay. As expected, the IC50s of the test compounds were much lower than
that of curcumin. In addition, compounds 5, 28, and 29 induced apoptosis in SGC-7901 cells more potently than
curcumin. Furthermore, compounds 5, 28, and 29 exhibited the ability to upregulate cleaved PARP and
downregulate expression of caspase-3 and Bcl-2 in SGC-7901 cells, while curcumin did not produce the same
effects. Moreover, compound 28 exhibited significant tumour inhibition in an SGC-7901-driven xenograft mouse
model. These results indicate that compound 28 has advantages over other compounds, as demonstrated by its
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anti-proliferative effect against gastrointestinal carcinoma cells lines and its significant induction of apoptosis and
regulation of apoptosis-related genes in SGC-7901 cells. The preliminary biological activity screening tests of our
novel series of curcumin derivatives also indicated that long-chain alkoxylated MACs might serve as potential
agents for the treatment of gastric cancer.
4. Material and Methods
4.1. Chemical synthesis
4.1.1. Materials and methods
Solvents were distilled and dried by standard methods. All chemicals were obtained from sigma-aldrich and
were used without further purification. All reactions were monitored by thin-layer chromatography (250 silica gel
1
60 F254 glass plates). H NMR and 13C NMR spectra were recorded on Bruker 600 MHz instruments, and the
chemical shifts were presented in terms of parts per million with TMS as the internal reference. Electron-spray
ionization mass spectra in positive mode (ESI-MS) data were obtained with a Bruker Esquire 3000+ spectrometer.
Chromatographic purification was carried out on Silica Gel 60 (E. Merck, 70-230 mesh).
4.1.2. General procedure for synthesis of X2
To a solution of p-hydroxybenzaldehyde or vanillin (1mmol) and anhydrous K2CO3 (3mmol.) in 20 ml DMF
was added requisite linear chain aliphatic dibromoalkanes (5mmol.). The reaction mixture was then stirred at 80°C
for 1h, cooled to 0 °C on an ice bath, quenched with 1N HCl and then extracted with CHCl3. The organic layer was
washed with water and dried over Na2SO4. Excess solvent was evaporated under reduced pressure and the resultant
solid left was purified by column chromatography over silica gel using EtOAc/Hexane as the eluent to afford
corresponding substituted aldehydes X2.(Yield: 67.5% and 75.3%)
4.1.3. General procedure for synthesis of X3
K2CO3 (7.58mmol) and desired amines (4.54mmol) were added to a stirred solution of substituted
benzaldehydes X2 (3.79mmol) in MeCN (15mL). The reaction mixture was then stirred at 80°C for 6h and
progress of reaction was monitored by TLC. After completion, it was cooled on ice bath and then extracted with
CHCl3. The organic layer was washed with water and dried over Na2SO4. Excess solvent was evaporated under
reduced pressure to obtain oily residue of corresponding amino substituted aldehydes X3, which were used for
further reaction. (Yield: 26.3%-69.7%)
4.1.4. General procedure for synthesis of 1-22
Cyclopentanone or Cyclohexanone (7.5mmol) was added to MeOH (5ml) with amino substituted
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benzaldehydes X3 (15mmol) inside. The solution was stirred for 15 min at room temperature followed by
dropwise addition of 20% (w/v) aq. NaOH solution. The reaction mixture was stirred at room temperature and
monitored by TLC. After completion of reaction, saturated solution of NH4Cl was added to the reaction mixture.
The formed precipitate was washed with water, cold methanol and finally with cold acetone. It was then dried and
recrystallized with ethanol/water system to obtain corresponding curcumin derivatives 1-22. (Yield: 15.4%-72.9%)
4.1.4.1 (2E,6E)-2,6-bis[3-methoxy-4-(3-morpholinopropoxy)benzylidene]cyclohexanone (1)
Yellow powder, m.p: 107.9–109.5 oC. 1H-NMR (600 MHz, CDCl3): δ (ppm) 7.726 (2H, s, -CH=×2 ), 7.067 (2H, d,
J = 8.4 Hz, Ar-H6×2), 7.007 (2H, s, Ar-H2×2), 6.909 (2H, d, J = 8.4 Hz, Ar-H5×2), 4.121 (4H, t, J = 6.6 Hz,
Ar-OCH2-×2), 3.872(6H, s, -OCH3×2), 3.754 (8H, t, J = 4.2 Hz, morpholine-H3,5×2), 2.927 (4H, t, J = 6 Hz,
cyclohexanone-H3,5), 2.624 (4H, t, J = 7.2 Hz, Ar-OCCCH2-×2), 2.582 (8H, t, J = 4.2 Hz, morpholine-H2,6×2),
2.070-2.093 (4H, m, Ar-OCCH2-×2), 1.810-1.823 (2H, m, cyclohexanone-H4). ESI-MS m/z: 621.6 (M+1)+, calcd
for C36H48N2O7: 620.8.
4.1.4.2 (2E,6E)-2-[4-(3-bromopropoxy)-3-methoxybenzylidene]-6-[3-methoxy-4-(3-morpholinopropoxy)benzyli
dene]cyclohexanone (2)
1
Yellow oil. H-NMR (600 MHz, CDCl3): δ (ppm) 7.734 (2H, s, -CH=×2 ), 7.021 (2H, d, J = 9.6 Hz, ArA-H6,
ArB-H6), 6.960 (2H, s, ArA-H2, ArB-H2), 6.916 (2H, d, J = 9.6 Hz, ArA-H5, ArB-H5), 4.118 (4H, t, J = 7.2 Hz,
ArA-OCH2-, ArB-OCH2-), 3.879 (6H, s, -OCH3×2), 3.744 (6H, m, morpholine-H3,5, Br-CH2-), 2.923 (4H, t, J = 6.6
Hz, cyclohexanone-H3,5), 2.596 (2H, t, J = 7.2 Hz, Ar-OCCCH2-), 2.532 (4H, t, J = 4.8 Hz, morpholine-H2,6),
2.356-2.389 (2H, m, Br-CCH2C-), 2.063-2.083 (2H, m, Ar-OCCH2-), 1.910-1.928 (2H, m, cyclohexanone-H4).
ESI-MS m/z: 615.4 (M+1)+, calcd for C32H40BrNO6: 614.6.
4.1.4.3 (2E,6E)-2,6-bis{4-[3-(dipropylamino)propoxy]-3-methoxybenzylidene}cyclohexanone(3)
1
Yellow oil. H-NMR (600 MHz, CDCl3): δ (ppm) 7.723 (2H, s, -CH=×2 ), 7.076 (2H, d, J = 8.4 Hz, Ar-H6×2),
7.000 (2H, s, Ar-H2×2), 6.897 (2H, d, J = 8.4 Hz, Ar-H5×2), 4.119 (4H, t, J = 5.4 Hz, -OCH2C-×2), 3.865 (6H, s,
-OCH3×2), 2.915 (4H, t, J = 6.6 Hz, cyclohexanone-H3,5), 2.651-2.717 (12H, m, NCH2-×6), 2.152 (4H, m,
-OCCH2C-×2), 1.611-1.649 (10H, m, -NCCH2-×4, cyclohexanone-H4), 0.939 (12H, t, J = 7.2 Hz, -CH3×4).
ESI-MS m/z: 649.7 (M+1)+, calcd for C40H60N2O5:648.9.
4.1.4.4 (2E,6E)-2,6-bis{3-methoxy-4-[3-(4-methylpiperazin-1-yl)propoxy]benzylidene}cyclohexanone(4)
Yellow powder, m.p: 94.5-96.1 oC. 1H-NMR (600 MHz, DMSO-d6): δ (ppm) 7.567 (2H, s, -CH=×2), 7.138 (2H, s,
Ar-H2×2), 7.058 (2H, d, J = 8.4 Hz, Ar-H6×2), 6.958 (2H, d, J = 8.4 Hz, Ar-H5×2), 4.009 (4H, t, J = 7.2 Hz,
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-OCH2C-×2), 3.782 (6H, s, -OCH3×2), 2.905 (4H, t, J = 6 Hz , cyclohexanone-H3,5), 2.624 (4H, t, J = 7.2 Hz,
-OCCCH2-×2), 2.483 (16H, s, piperazine–H2,3,5,6×2), 2.154 (6H, s, NCH3×2), 1.812-1.877 (4H, m,
-OCCH2CN-×2), 1.148-1.162 (2H, m, cyclohexanone-H4). ESI-MS m/z: 647.7 (M+1)+, calcd for
C38H54N4O5:646.8.
4.1.4.5 (2E,6E)-2,6-bis{4-[3-(diethylamino)propoxy]-3-methoxybenzylidene}cyclohexanone(5)
o
1
Yellow powder, m.p: 294.2-295.6 C. H-NMR (600 MHz, DMSO-d6): δ (ppm) 7.579 (2H, s, -CH=×2 ), 7.122
(2H, s, Ar-H2×2), 7.103 (2H, d, J = 8.4 Hz, Ar-H6×2), 7.003 (2H, d, J = 8.4 Hz, Ar-H5×2), 4.021 (4H, t, J = 6 Hz,
-OCH2C-×2), 3.790 (6H, s, -OCH3×2), 2.890 (4H, t, J = 5.4 Hz, cyclohexanone-H3,5), 2.419-2.454 (12H, m,
-NCH2-×6), 1.792-1.814 (6H, m, -OCCH2C-×2, cyclohexanone-H4), 0.926 (12H, t, J = 6.6 Hz, -NCCH3×4).
13C-NMR (600 MHz, DMSO-d6): δ (ppm) 188.512, 149.012×2, 148.685×2, 135.860×2, 134.237×2, 128.103×2,
123.823×2, 114.412×2, 112.629×2, 66.548×2, 55.629×2, 48.626×2, 46.418×4, 27.901×2, 26.613×2, 22.509,
11.768×4. ESI-MS m/z: 593.7 (M+1)+, calcd for C36H52N2O5: 592.8.
4.1.4.6 (2E,6E)-2,6-bis{4-[3-(1H-imidazol-1-yl)propoxy]-3-methoxybenzylidene}cyclohexanone(6)
Yellow oil. 1H-NMR (600 MHz, DMSO-d6): δ (ppm) 7.617 (2H, s, imidazole-H2×2 ), 7.588 (2H, s, Ar-CH=×2),
7.158-7.195 (4H, m, Ar-H2,6×2), 7.106 (2H, d, J = 7.8 Hz, imidazole-H5×2), 7.009 (2H, d, J = 8.4 Hz, Ar-H5×2),
6.891 (2H, d, J = 7.8 Hz, imidazole-H4×2), 4.136 (8H, t, J = 6.6 Hz,-OCH2CCH2-×2), 3.810 (6H, s, -OCH3×2),
2.901 (4H, t, J = 5.4 Hz, cyclohexanone-H3,5), 2.169-2.191 (4H, m, -OCCH2C-), 1.179-1.701 (2H, m,
cyclohexanone-H4). ESI-MS m/z: 583.7 (M+1)+, calcd for C34H38N4O5:582.7.
4.1.4.7 (2E,6E)-2,6-bis{3-methoxy-4-[3-(piperidin-1-yl)propoxy]benzylidene}cyclohexanone(7)
Yellow powder, m.p: 192.3-193.4 oC. 1H-NMR (600 MHz, DMSO-d6): δ (ppm) 7.583(2H, s, -CH=×2), 7.132 (2H,
s, Ar-H2×2), 7.113 (2H, d, J = 8.4 Hz, Ar-H6×2), 7.025 (2H, d, J = 8.4 Hz, Ar-H5×2), 4.031 (4H, t, J = 6.6 Hz,
OCH2×2), 3.798(6H, s, –OCH3×2), 2.909 (4H, t, J = 6 Hz , cyclohexanone-H3,5), 2.384-2.413 (12H, m, NCH2×6),
2.128-2.144 (4H, m, OCCH2C-×2), 1.848-1.865 (14H, m, piperidine-H3,4,5×2, cyclohexanone-H4). ESI-MS m/z:
617.9 (M+1)+, calcd for C38H52N2O5:616.8.
4.1.4.8 (2E,6E)-2,6-bis[4-(3-bromopropoxy)-3-methoxybenzylidene]cyclohexanone(8)
Yellow powder, m.p: 123.7-124.4 oC. 1H-NMR (600 MHz, DMSO-d6): δ (ppm) 7.589 (2H, s, -CH=×2), 7.151 (2H,
s, Ar-H2×2), 7.130 (2H, d, J = 9 Hz, Ar-H6×2), 7.025 (2H, d, J = 9 Hz, Ar-H5×2), 4.118 (4H, t, J = 6 Hz, OCH2×2),
3.810 (6H, s, –OCH3×2), 3.661 (4H, t, J = 6.6 Hz, BrCH2-×2), 2.909 (4H, t, J = 4.8 Hz, cyclohexanone-H3,5),
2.255-2.267 (4H, m, BrCCH2-×2), 1.690-1.710 (2H, m, cyclohexanone-H4). ESI-MS m/z: 608.9 (M+1)+, calcd for
9

ACCEPTED MANUSCRIPT
C28H32Br2O5: 608.3.
4.1.4.9 (2E,6E)-2-[4-(3-bromopropoxy)-3-methoxybenzylidene]-6-{4-[3-(diethylamino)propoxy]-3-methoxy
benzylidene}cyclohexanone(9)
1
Yellow oil. H-NMR (600 MHz, DMSO-d6): δ (ppm) 7.542 (2H, s, ArA-CH=C-, ArB-CH=C-), 7.196 (2H, s,
ArA-H2, ArB-H2), 7.142 (2H, d, J = 8.4 Hz, ArA-H6, ArB-H6), 6.975 (2H, d, J = 8.4 Hz, ArA-H5, ArB-H5), 4.022 (4H,
m, ArA–OCH2C-, ArB–OCH2C-), 3.911 (6H, s, -OCH3×2), 3.675 (2H, t, J = 6 Hz, BrCH2-), 3.108-3.125 (4H, m,
-NCH2C×2), 2.930 (4H, t, J = 6.6 Hz, cyclohexanone-H3,5), 2.398 (2H, t, J = 6 Hz, ArB-OCCCH2-), 2.162-2.184
(2H, m, BrCCH2-), 2.013-2.025 (2H, m, ArB-OCCH2C-), 1.720-1.734 (2H, m, cyclohexanone-H4), 1.189 (6H, s,
-NCCH3×2). ESI-MS m/z: 601.4 (M+1)+, calcd for C32H42BrNO5:600.6.
4.1.4.10 (2E,6E)-2-[4-(3-bromopropoxy)-3-methoxybenzylidene]-6-{3-methoxy-4-[3-(piperidin-1-yl)propoxy]
benzylidene}cyclohexanone(10)
Yellow oil. 1H-NMR (600 MHz, DMSO-d6): δ (ppm) 7.571 (2H, s, ArA-CH=C-, ArB-CH=C-), 7.142-7.160 (4H, m,
ArA-H2,6, ArB-H2,6), 7.028 (2H, d, J = 8.4 Hz, ArA-H5, ArB-H5), 4.085 (2H, t, J = 6 Hz, ArA-OCH2C-), 3.992 (2H, t,
J = 6.6 Hz, ArB-OCH2C-), 3.811 (6H, s, -OCH3×2), 3.789 (2H, t, J = 6 Hz , BrCH2-), 2.884 (4H, t, J = 6 Hz,
cyclohexanone-H3,5), 2.301-2.367 (6H, m, NCH2-×2), 1.870-1.893 (2H, m, -OCCH2CBr), 1.823-1.859 (2H, m,
ArB-OCCH2C-), 1.447-1.484 (8H, m, piperidine-H3,4,5, cyclohexanone-H4). ESI-MS m/z: 613.4 (M+1)+, calcd for
C33H42BrNO5: 612.6.
4.1.4.11 (2E,6E)-2-[4-(3-bromopropoxy)-3-methoxybenzylidene]-6-{3-methoxy-4-[3-(4-methylpiperazin-1-yl)
propoxy]benzylidene}cyclohexanone (11)
Yellow oil.1H-NMR (600 MHz, DMSO-d6): δ (ppm) 7.576 (2H, s, ArA-CH=C-, ArB-CH=C-), 7.512 (2H, d, J = 8.4
Hz, ArA-H6, ArB-H6), 7.388 (2H, s, ArA-H2, ArB-H2), 7.168 (2H, d, J = 8.4 Hz, ArA-H5, ArB-H5), 4.103 (2H, t, J =
6.6 Hz, ArA-OCH2C-), 4.027 (2H, t, J = 7.2 Hz, ArB-OCH2C-), 3.828(6H, s, -OCH3×2), 3.754 (2H, t, J = 6.6 Hz ,
BrCH2), 2.539 (4H, t, J = 6 Hz, cyclohexanone-H3,5), 2.402 (2H, t, J = 6.6 Hz, piperazine-CH2-), 2.140 (8H, s,
piperazine-H2,3,5,6), 1.972-1.986 (5H, m, -OCCH2CBr-, NCH3), 1.878-1.901 (2H, m, ArB-OCCH2C-), 1.161-1.185
(2H, m, cyclohexanone-H4). ESI-MS m/z: 628.5 (M+1)+, calcd for C33H43BrN2O5: 627.6.
4.1.4.12 (2E,6E)-2,6-bis[4-(2-bromoethoxy)-3-methoxybenzylidene]cyclohexanone(12)
Yellow powder, m.p: 199.8-200.9 oC.1H-NMR (600 MHz, DMSO-d6): δ(ppm) 7.592 (2H, s, -CH=×2 ), 7.167 (2H,
s, Ar-H2×2), 7.122 (2H, d, J = 8.4 Hz, Ar-H6×2), 7.054 (2H, d, J = 8.4 Hz, Ar-H5×2), 4.355 (4H, t, J = 5.4 Hz,
-OCH2-×2), 3.822 (6H, s, -OCH3×2), 3.789(4H, t, J = 6 Hz, -CH2Br×2), 2.910 (4H, t, J = 6 Hz,
10

ACCEPTED MANUSCRIPT
cyclohexanone-H3,5), 1.722-1.740 (2H, m, cyclohexanone-H4). ESI-MS m/z: 581.0 (M+1)+, calcd for C26H28Br2O5:
580.0.
4.1.4.13 (2E,6E)-2,6-bis[4-(2-bromoethoxy)benzylidene]cyclohexanone(13)
Yellow powder, m.p: 101.3-102.7oC.1H-NMR (600 MHz, DMSO-d6): δ (ppm) 7.582 (2H, s, -CH=×2), 7.519 (4H,
d, J = 8.4 Hz, Ar-H2,6×2), 7.044 (4H, d, J = 8.4 Hz, Ar-H3,5×2), 4.380 (4H, t, J = 5.4 Hz, -OCH2CBr-×2), 3.822(4H,
t, J = 5.4 Hz, -OCCH2Br-×2), 2.876 (4H, t, J = 4.8 Hz , cyclohexanone-H3,5), 1.128-1.171 (2H, m,
cyclohexanone-H4). ESI-MS m/z: 521.3 (M+1)+, calcd for C24H24Br2O3: 520.3.
4.1.4.14 (2E,5E)-2-{4-[3-(1H-imidazol-1-yl)propoxy]-3-methoxybenzylidene}-5-[4-(3-bromopropoxy)-3-metho
xybenzylidene]cyclopentanone(14)
Yellow oil.1H-NMR (600 MHz, DMSO-d6): δ(ppm) 7.699 (1H, s, imidazole-H2 ), 7.399 (2H, s, Ar-CH=C-×2),
7.295-7.229 (5H, m, Ar-H2,6×2, imidazole-H5), 7.059 (2H, d, J = 8.4 Hz, Ar-H5×2), 6.936 (1H, d, J = 7.8 Hz,
imidazole-H4), 4.140-4.163 (6H, m,-OCH2CCBr-,-OCH2CCH2N-), 3.869 (6H, s, -OCH3×2), 3.671 (2H, t, J = 6 Hz,
-OCCCH2Br), 3.094 (4H, s, cyclopentanone-H4,5), 2.197-2.216 (4H, m, -OCCH2CBr, -OCCH2CN-). ESI-MS m/z:
582.3 (M+1)+, calcd for C30H33BrN2O5: 581.5.
4.1.4.15 (2E,5E)-2,5-bis[4-(3-bromopropoxy)-3-methoxybenzylidene]cyclopentanone(15)
Yellow powder, m.p: 137.6-139.1oC.1H-NMR (600 MHz, CDCl3): δ (ppm) 7.542 (2H, s, -CH=×2), 7.227 (2H, d, J
= 8.4 Hz, Ar-H6×2), 7.139 (2H, s, Ar-H2×2), 6.975 (2H, d, J = 8.4 Hz, Ar-H5×2), 4.220 (4H, t, J = 6 Hz,
-OCH2-×2), 3.910 (6H, s, -OCH3×2), 3.639 (4H, t, J = 6 Hz, -CH2Br×2), 3.115 (4H, s, cyclopentanone-H3,4),
2.377-2.417 (4H, m, -OCCH2CBr×2). ESI-MS m/z: 595.1 (M+1)+, calcd for C27H30Br2O5: 594.3.
4.1.4.16 (2E,5E)-2,5-bis{4-[3-(1H-imidazol-1-yl)propoxy]-3-methoxybenzylidene}cyclopentanone(16)
Yellow powder, m.p: 127.3-129.2oC.1H-NMR (600 MHz, CDCl3): δ (ppm) 7.538 (2H, s, imidazole-H2×2 ), 7.512
(2H, s, Ar-CH=×2 ), 7.187 (2H, d, J = 7.8 Hz, Ar-H6×2), 7.147 (2H, s, Ar-H2×2), 7.060 (2H, d, J = 8.4 Hz,
imidazole-H5×2), 6.937 (2H, d, J = 7.8 Hz, Ar-H5×2), 6.875 (2H, d, J = 8.4 Hz, imidazole-H4×2), 4.230 (4H, t, J =
6 Hz , -OCH2-×2), 3.990 (4H, t, J = 6 Hz, -OCCCH2-×2), 3.934 (6H, s, -OCH3×2), 3.111 (4H, s,
cyclopentanone-H3,4), 2.313-2.271 (4H, m, -OCCH2-×2). ESI-MS m/z: 648.92 (M-1)-. ESI-MS m/z:569.8 (M+1)+,
calcd for C33H36N4O5: 568.7.
4.1.4.17 (2E,5E)-2,5-bis[3-methoxy-4-(3-morpholinopropoxy)benzylidene]cyclopentanone(17)
Yellow powder, m.p: 176.4-177.8oC 1H-NMR (600 MHz, DMSO-d6): δ(ppm) 7.375 (2H, s, -CH=×2 ), 7.253 (2H,
11

ACCEPTED MANUSCRIPT
d, J = 7.2 Hz, Ar-H6×2), 7.215 (2H, s, Ar-H2×2), 7.059 (2H, d, J = 7.2 Hz, Ar-H5×2), 4.049 (4H, t, J = 6.6 Hz,
-OCH2-×2), 3.809 (6H, s, -OCH3×2), 3.554 (8H, t, J = 4.2 Hz, morpholine-H3,5×2), 3.079 (4H, s,
cyclopentanone-H3,4), 2.522 (4H, t, J = 6.6 Hz, Ar-OCCCH2-×2), 2.483 (8H, t, J = 4.2 Hz, morpholine-H2,6×2),
1.132-1.148 (4H, m, -OCCH2C-×2). ESI-MS m/z: 607.7 (M+1)+, calcd for C35H46N2O7: 606.8.
4.1.4.18 (2E,5E)-2,5-bis{3-methoxy-4-[3-(piperidin-1-yl)propoxy]benzylidene}cyclopentanone(18)
Yellow powder, m.p: 198.3-200.2 oC .1H-NMR (600 MHz, DMSO-d6): δ(ppm) 7.402 (2H, s, -CH=×2 ), 7.287 (2H,
s, Ar-H2×2), 7.270 (2H, d, J = 8.4 Hz, ArA-H6×2), 7.085 (2H, d, J = 8.4 Hz, Ar-H5×2), 4.079 (4H, t, J = 6 Hz,
-OCH2-), 3.835 (6H, s, -OCH3×2), 3.100 (4H, s, cyclopentanone-H3,4), 2.560-2.702 (12H, m, NCH2-×6),
1.976-1.995 (4H, m, -OCCH2C-×2), 1.442-1.586 (12H, m, piperidine-H3,4,5×2). ESI-MS m/z: 603.7 (M+1)+, calcd
for C37H50N2O5: 602.8.
4.1.4.19
(2E,5E)-2-[4-(3-bromopropoxy)-3-methoxybenzylidene]-5-{3-methoxy-4-[3-(piperidin-1-yl)propoxy]
benzylidene}cyclopentanone(19)
Yellow oil.1H-NMR (600 MHz, DMSO-d6): δ(ppm) 7.410 (2H, s, ArA-CH=, ArB-CH=), 7.340-7.385 (4H, m,
ArA-H2,6, ArB-H2,6), 6.980 (2H,d, J = 8.4 Hz, ArA-H5, ArB-H5), 4.162 (2H, t, J = 5.4 Hz, ArA-OCH2C-), 4.089 (2H, t,
J = 6 Hz, ArB-OCH2C-), 3.870 (6H, s, -OCH3×2), 3.573 (2H, t, J = 5.4 Hz, -CH2Br), 3.110 (4H, s,
cyclopentanone-H3,4), 2.370-2.412 (6H, m, NCH2-×3), 2.245-2.307 (2H, m, -OCCH2CBr), 1.967-1.980 (2H, m,
piperidine-CCH2-), 1.321-1.413 (6H, m, piperidine-H3,4,5). ESI-MS m/z: 599.3 (M+1)+, calcd for C32H40BrNO5:
598.6.
4.1.4.20 (2E,5E)-2,5-bis[4-(2-bromoethoxy)-3-methoxybenzylidene]cyclopentanone(20)
Yellow powder, m.p: 155.2-156.9oC. 1H-NMR (600 MHz, DMSO-d6): δ(ppm) 7.406 (2H, s, -CH=×2 ), 7.307 (2H,
s, Ar-H2×2), 7.265 (2H, d, J = 8.4 Hz, ArA-H6×2), 7.097 (2H, d, J = 8.4 Hz, Ar-H5×2), 4.376 (4H, t, J = 5.4 Hz,
-OCH2-×2), 3.851 (6H, s, -OCH3×2), 3.820 (4H, t, J = 5.4 Hz, -CH2Br×2), 3.100 (4H, s, cyclopentanone-H3,4).
ESI-MS m/z: 567.5 (M+1)+, calcd for C25H26Br2O5: 566.3.
4.1.4.21 (2E,5E)-2,5-bis[4-(2-bromoethoxy)benzylidene]cyclopentanone(21)
Yellow powder, m.p: 206.3-207.6oC. 1H-NMR (600 MHz, DMSO-d6): δ(ppm) 7.662 (4H, d, J = 9 Hz, Ar-H2,6×2),
7.401(2H, s, -CH=×2,), 7.086 (4H, d, J =9 Hz, Ar-H3,5×2), 4.402 (4H, t, J = 5.4 Hz, -OCH2CBr×2), 3.833 (4H, t, J
= 5.4 Hz, -OCCH2Br×2), 3.063 (4H, s, cyclopentanone-H3,4). ESI-MS m/z: 507.1 (M+1)+, calcd for C23H22Br2O3:
506.2.
12

ACCEPTED MANUSCRIPT
4.1.4.22 (1E,4E)-1,5-bis[3-methoxy-4-(3-morpholinopropoxy)phenyl]penta-1,4-dien-3-one(22)
Yellow oil. 1H-NMR (600 MHz, DMSO-d6): δ(ppm) 7.688 (2H, d, J = 15.6 Hz , Ar-CH=C-×2 ), 7.136-7.153 (4H,
m, Ar-H2,6×2), 7.022-7.043 (4H, m,, Ar-C=CH-×2, Ar-H5×2), 4.058 (4H, t, J = 6 Hz, -OCH2-×2), 3.842 (6H, s,
-OCH3×2), 3.564 (8H, t, J = 5.4 Hz , morpholine-H3,5×2), 2.409 (4H, t, J = 6 Hz, -OCCCH2-×2), 2.360 (8H, t, J =
5.4 Hz, morpholine-H2,6×2), 1.831-1.904 (4H, m, -OCCH2C×2). ESI-MS m/z: 581.8 (M+1)+, calcd for
C33H44N2O7: 580.7.
4.1.5. General procedure for synthesis of X4
Amino substituted benzaldehydes X3 were dissolved in acetone and followed by dropwise addition of 20%
(w/v) aq. NaOH solution. The mixture was stirred at room temperature for 3h. Excess acetone was evaporated
under reduced pressure and the residue was extracted with ethyl acetate and the organic layer washed with brine,
dried over anhydrous magnesium sulphate, and concentrated under vacuum. The solid was purified by
chromatography over silica gel using ethyl acetate/petroleum ether as the eluent to give compounds X4.
4.1.6. General procedure for synthesis of 23-37
A reaction mixture of the compound X4 (1.0 equiv), substituted benzaldehydes (1.0 equiv) and 20% (w/v)
aq. NaOH (0.1 equiv) in MeOH was stirred at room temperature and monitored by TLC. After completion of
reaction, saturated solution of NH4Cl was added to the reaction mixture. The formed precipitate was washed with
water, cold methanol and finally with cold acetone. It was then dried and recrystallized with ethanol/water system
to obtain corresponding curcumin derivatives 23-37. (Yield:24.3%-76.8%)
4.1.6.1 (1E,4E)-1-(2-chlorophenyl)-5-[3-methoxy-4-(3-morpholinopropoxy)phenyl]penta-1,4-dien-3-one(23)
Yellow powder, m.p: 135.6-137.4oC.1H-NMR (600 MHz, CDCl3): δ (ppm) 8.110 (1H, d, J = 15.6 Hz, ArB-CH=C-),
7.714 (1H, d, J = 15.6 Hz, ArA-CH=C-), 7.528 (1H, d, J = 7.2 Hz, ArB-H3), 7.384 (1H, d, J = 7.2 Hz, ArB-H6),
7.302-7.329 (2H, m, ArA-H2 ArB-H5), 7.133-7.211 (3H, m, ArA-H5,6, ArB-H4), 7.054 (1H, d, J = 15.6 Hz,
ArA-C=CH-), 6.969 (1H, d, J = 15.6 Hz , ArB-C=CH-), 4.162 (2H, t, J = 7.2 Hz, -OCH2-), 3.913 (3H, s, -OCH3),
3.810 (4H, s, morpholine-H3,5), 2.230 (2H, t, J = 7.2 Hz, -OCCCH2-), 2.309 (4H, s, morpholine-H2,6), 1.405-1.444
(2H, m, -OCCH2C-). ESI-MS m/z: 441.95 (M-1)-. ESI-MS m/z: 443.0 (M+1)+, calcd for C25H28ClNO4: 442.0.
4.1.6.2 (1E,4E)-1-(2-fluoro-4-methoxyphenyl)-5-[3-methoxy-4-(3-morpholinopropoxy)phenyl]penta-1,4-dien-
3-one(24)
Red oil.1H-NMR (600 MHz, CDCl3): δ(ppm) 7.719 (1H, d, J = 16.2 Hz, ArB-CH=C-), 7.670 (1H, d, J = 16.2 Hz,
ArA-CH=C-), 7.542 (1H, d, J = 8.4 Hz, ArB-H6), 7.178 (1H, d, J = 8.4Hz, ArA-H6), 7.135 (1H, s, ArA-H2),
13

ACCEPTED MANUSCRIPT
7.075(1H, d, J = 16.2 Hz, ArA-C=CH-), 6.900-6.947 (3H, m, ArB-H3, ArA-H5, ArB-C=CH-), 6.743 (1H, d, J = 8.4
Hz, ArB-H5), 4.155 (2H, t, J = 6.6 Hz, -OCH2-), 3.915 (3H, s, ArA-OCH3,), 3.845 (3H, s, ArB-OCH3,), 3.806 (4H, s,
morpholine-H3,5), 2.505 (2H, t, J = 6.6 Hz, -OCCCH2-), 2.214 (4H, s, morpholine-H2,4), 1.251-1.313 (2H, m,
-OCCH2C-). ESI-MS m/z: 456.6 (M+1)+, calcd for C26H30FNO5: 455.5.
4.1.6.3 (1E,4E)-1-(3,4-dimethoxyphenyl)-5-[3-methoxy-4-(3-morpholinopropoxy)phenyl]penta-1,4-dien-3-one
(25)
Yellow oil .1H-NMR (600 MHz, CDCl3): δ (ppm) 7.707 (1H, d, J = 15.6 Hz , ArB-CH=C- ), 7.531 (1H, d, J = 15.6
Hz , ArA-CH=C-), 7.198-7.173 (4H, m, ArB-H2,ArB-H6, ArA-H2, ArA-H6,), 6.987-6.870 (4H, m, ArB-H5, ArA-H5,
ArA-C=CH-, ArB-C=CH-), 4.295 (2H, t, J = 7.2 Hz, -OCH2-), 4.081 (6H, s, ArA-OCH3, ArB-3-OCH3), 4.070 (3H, s,
ArB-4-OCH3), 3.921 (4H, s, morpholine-H3,5), 2.520 (2H, t, J =7.2 Hz, -OCCCH2-), 2.293 (4H, s, morpholine-H2,6),
1.386-1.437 (2H, m, -OCCH2-). ESI-MS m/z: 467.5 (M+1)+, calcd for C27H33NO6: 467.6.
4.1.6.4
(1E,4E)-1-[3-methoxy-4-(3-morpholinopropoxy)phenyl]-5-(2,4,6-trimethoxyphenyl)penta-1,4-dien-3-
one(26)
1
Yellow powder, m.p: 125.4-126.7oC. H-NMR (600 MHz, DMSO-d6): δ(ppm) 7.995 (1H, d, J = 15.6 Hz ,
ArB-CH=C ), 7.534 (1H, d, J = 15.6 Hz, ArA-CH=C), 7.393 (1H, s, ArA-H2), 6.947-7.205 (3H, m, ArA-H5, ArA-H6,
ArA-C=CH-), 6.655 (1H, s, J = 15.6 Hz, ArB-C=CH-), 6.213 (2H, s, ArB-H3, ArB-H5), 4.038 (2H, t, J = 7.2 Hz,
-OCH2-), 3.906 (6H, s, ArB-2,6-OCH3), 3.854 (3H, s, ArA-OCH3), 3.833 (3H, s, ArB-4-OCH3), 3.560 (4H, s,
morpholine-H3,5), 2.393 (2H, t, J = 7.2 Hz, -OCCCH2-), 2.350 (4H, s, morpholine-H2,6), 1.860-1.884 (2H, m,
-OCCH2-). ESI-MS m/z: 498.3 (M+1)+, calcd for C28H35NO7: 497.6.
4.1.6.5 (1E,4E)-1-mesityl-5-[3-methoxy-4-(3-morpholinopropoxy)phenyl]penta-1,4-dien-3-one(27)
White powder, m.p: 137.5-139.1oC. 1H-NMR (600 MHz, CDCl3): δ(ppm) 7.878 (1H, d, J = 16.2 Hz, ArB-CH=C-),
7.649 (1H, d, J = 16.2 Hz, ArA-CH=C-), 7.123 (1H, s, ArA-H2), 6.884-6.917 (5H, m, ArA-H5,6, ArB-H3,5
,
ArA-C=CH-), 6.713 (1H, d, J = 16.2 Hz, ArB-C=CH-), 4.153 (2H, t, J = 6 Hz , -OCH2-), 3.909 (3H, s, -OCH3 ),
3.838 (4H, s, morpholine-H3,5), 2.376 (6H, s, ArB-2-CH3, ArB-6-CH3), 2.298 (3H, s, ArB-4-CH3), 2.260 (2H, t, J =
6 Hz, -OCCCH2-), 2.206 (4H, s, morpholine-H2,6), 2.035-2.054 (2H, m, -OCCH2C-). ESI-MS m/z: 450.5(M+1)+,
calcd for C28H35NO4: 449.6.
4.1.6.6 (1E,4E)-1-[3-methoxy-4-(3-morpholinopropoxy)phenyl]-5-(2-methoxyphenyl)penta-1,4-dien-3-one (28)
Yellow powder, m.p: 113.2-124.1oC. 1H-NMR (600 MHz, CDCl3): δ(ppm) 8.040 (1H, d, J = 15.6 Hz ,
ArB-CH=C-), 7.652 (1H, d, J = 15.6 Hz, ArA-CH=C-), 7.585 (1H, d, J =7.8 Hz, ArB-H6), 7.323-7.349 (1H, m,
14

ACCEPTED MANUSCRIPT
ArB-H4), 7.112-7.163 (3H, m, ArA-H2, ArA-H6, ArB-H3), 6.882-6.965 (4H, m, ArA-H5, ArB-H5, ArA-C=CH-,
ArB-C=CH-), 4.110 (2H, t, J = 6 Hz,-OCH2C-), 3.890 (3H, s, ArA-OCH3), 3.862 (3H, s, ArB-OCH3), 3.703 (4H, s,
morpholine-H3,5), 2.530 (2H, t, J = 6 Hz, -OCCCH2-), 2.461 (4H, s, morpholine-H2,6), 2.012-2.054 (2H, m,
-OCCH2C-). 13C-NMR (600 MHz, CDCl3): δ (ppm) 188.802, 158.108, 150.270, 149.075×2, 142.492, 137.745,
131.149, 128.266, 127.485, 125.715, 123.265, 122.444, 120.266, 112.184, 110.729, 110.106, 66.694, 66.366×4,
55.539, 53.162×2, 25.632. ESI-MS m/z: 438.3(M+1)+, calcd for C26H31NO5: 437.5.
4.1.6.7 (1E,4E)-1-(2,3-dimethoxyphenyl)-5-[3-methoxy-4-(3-morpholinopropoxy)phenyl]penta-1,4-dien-3-one
(29)
Yellow oil. 1H-NMR (600 MHz, DMSO-d6): δ(ppm) 8.016(1H, d, J = 16.2 Hz, ArB-CH=C-), 7.798(1H, d, J = 16.2
Hz ,ArA-CH=C-), 7.383 (1H, d, J = 7.8 Hz, ArB-H6), 7.224 (1H, s, ArA-H2), 7.099-7.112 (1H, m, ArB-H5),
7.040-7.099 (4H, m, ArA-H5,6, ArA-C=CH-, ArB-C=CH-), 6.925 (1H, t, J = 7.8 Hz, ArB-H4), 4.098 (2H, t, J = 6.6
Hz, -OCH2C-), 3.838 (6H, s, ArA-3-OCH3, ArB-3-OCH3), 3.796 (3H, s, ArB-2-OCH3), 3.561 (4H, s,
morpholine-H3,5), 2.407 (2H, t, J = 6.6 Hz, -OCCCH2-), 2.380 (4H, s, morpholine-H2,6), 1.901-1.940 (2H, m,
-OCCH2C-). 13C-NMR (600 MHz, DMSO-d6): δ (ppm) 188.712, 153.022, 152.700, 148.797×2, 147.487, 147.133,
141.769, 126.878, 124.230×2, 121.015, 120.553, 120.380, 119.136, 114.179, 112.134, 69.710, 66.451×2, 65.577×2,
62.299, 61.313, 56.035, 55.884, 27.243. ESI-MS m/z: 468.7 (M+1)+, calcd for C27H33NO6: 467.6.
4.1.6.8 (1E,4E)-1-[4-(3-bromopropoxy)-3-methoxyphenyl]-5-(2-methoxyphenyl)penta-1,4-dien-3-one(30)
Yellow oil. 1H-NMR (600 MHz, DMSO-d6): δ(ppm) 7.958 (1H, d, J = 15.6 Hz , ArB-CH=C- ), 7.81 (1H, d, J = 7.2
Hz, ArB-H6), 7.746 (1H, d, J = 15.6 Hz , ArA-CH=C-), 7.305-7.436 (2H, m, ArA-H2, ArB-H4), 7.009-7.175 (6H, m,
ArA-H5,6, ArB-H3,5, ArA-C=CH-, ArB-C=CH-), 4.123 (2H, t, J = 6 Hz, -OCH2-), 3.886 (3H, s, ArB-OCH3), 3.849
(3H, s, ArA-OCH3), 3.658 (2H, t, J = 6 Hz, BrCH2-), 1.972-1.990 (2H, m, BrCCH2C-). ESI-MS m/z: 431.9(M+1)+,
calcd for C22H23BrO4: 431.3.
4.1.6.9 (1E,4E)-1-[4-(2-bromoethoxy)-3-methoxyphenyl]-5-(2-methoxyphenyl)penta-1,4-dien-3-one(31)
Yellow powder, m.p: 116.6-117.9oC. 1H-NMR (600 MHz, CDCl3): δ(ppm) 8.068 (1H, d, J = 16.2 Hz ,
ArB-CH=C- ), 7.671 (1H, d, J = 15.6 Hz , ArA-CH=C-), 7.619 (1H, d, J =7.2 Hz, ArB-H6), 7.364-7.390 (1H, m,
ArB-H4), 7.132-7.199 (2H, m, ArA-H2,6), 6.909-7.005 (5H, m, ArA-H5, ArB-H3,5, ArA-C=CH-, ArB-C=CH-), 4.378
(2H, t, J = 6.6 Hz, -OCH2-), 3.935 (3H, s, ArB-OCH3), 3.922 (3H, s, ArA-OCH3), 3.681 (2H, t, J =6.6 Hz, Br-CH2-).
ESI-MS m/z: 418.0(M+1)+, calcd for C21H21BrO4: 417.3.
4.1.6.10 (1E,4E)-1-[4-(tert-butyl)phenyl]-5-[3-methoxy-4-(3-morpholinopropoxy)phenyl]penta-1,4-dien-3-one
15

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(32)
1
Yellow oil. H-NMR (600 MHz, DMSO-d6): δ(ppm) 7.705 (1H, d, J = 15.6 Hz, ArB-CH=C-), 7.665 (1H, d, J =
15.6 Hz, ArA-CH=C-), 7.532 (2H, d, J = 8.4 Hz, ArB-H2,6), 7.400 (2H, d, J = 8.4 Hz, ArB-H3,5), 7.147 (1H, d, J =
8.4 Hz, ArA-H6), 7.122 (1H, s, ArA-H2), 7.047 (1H, d, J = 15.6 Hz, ArB-C=CH-), 6.936 (1H, d, J = 15.6 Hz,
ArA-C=CH-), 6.881 (1H, d, J = 8.4 Hz, ArA-H5), 4.106 (2H, t, J = 6.6 Hz, -OCH2C-), 3.888 (3H, s, -OCH3), 3.715
(4H, s, morpholine-H3,5), 2.560 (2H, t, J = 7.2 Hz, -OCCCH2-), 2.495 (4H, s, morpholine-H2,6), 2.023-2.069 (2H,
m, -OCCH2C-), 1.310 (9H, s,–CCH3×3). ESI-MS m/z: 464.7 (M+1)+, calcd for C29H37NO4: 463.6.
4.1.6.11 (1E,4E)-1-[3-methoxy-4-(3-morpholinopropoxy)phenyl]-5-[2-(trifluoromethyl)phenyl]penta-1,4-dien-3
-one(33)
1
Yellow oil. H-NMR (600 MHz, DMSO-d6): δ(ppm) 8.125 (1H, d, J = 15.6 Hz, ArB-CH=C-), 7.808 (1H, d, J =
15.6 Hz, ArA-CH=C-), 7.387-7.719 (4H, m, ArA-H2, ArB-H3,5,6), 6.832-7.135 (4H, m, ArA-H5,6, ArB-H4,
ArA-C=CH-), 6.683 (1H, d, J = 15.6 Hz, ArB-C=CH-), 4.218 (2H, d, J = 6.6 Hz, -OCH2C-), 3.827 (3H, s, -OCH3,),
3.792 (4H, s, morpholine-H3,5), 2.360 (2H, t, J = 6.6 Hz, -OCCCH2-), 2.301 (4H, s, morpholine-H2,6), 1.821-1.909
(2H, m, -OCCH2C-). ESI-MS m/z: 476.4 (M+1)+, calcd for C26H28F3NO4: 475.5.
4.1.6.12
(2E,6E)-2-(2,3-dimethoxybenzylidene)-6-[3-methoxy-4-(3-morpholinopropoxy)benzylidene]cyclohex
anone(34)
Red oil. 1H-NMR (600 MHz, DMSO-d6): δ(ppm) 7.712 (1H, s, ArB-CH= ), 7.682 (1H, s, ArA-CH= ), 7.512-7.571
(2H, m, ArA-H6, ArB-H6), 7.394 (1H, s, ArA-H2), 7.055-7.161 (3H, m, ArA-H5, ArB-H4,5), 4.106 (2H, t, J = 6.6 Hz,
-OCH2-), 3.826 (9H, s, -OCH3×3), 3.560 (4H, s, morpholine-H3,5), 2.898 (4H, t, J = 6.6 Hz, cyclohexanone-H3,5),
2.410 (2H, t, J =6.6 Hz, -OCCCH2-), 2.352 (4H, s, morpholine-H2,6), 1.891-1.914 (2H, m, -OCCH2C-),
1.558-1.650 (2H, m, cyclohexanone-H4). ESI-MS m/z: 508.3 (M+1)+, calcd for C30H37NO6: 507.6.
4.1.6.13
(2E,6E)-2-(3,4-dimethoxybenzylidene)-6-[3-methoxy-4-(3-morpholinopropoxy)benzylidene]cyclohex
anone(35)
1
Yellow oil. H-NMR (600 MHz, CDCl3): δ(ppm) 7.732 (2H, s, -CH=×2), 7.012-7.112 (4H, m, ArA-H2, ArA-H6,
ArB-H2, ArB-H6), 6.915 (1H, d, J = 7.2 Hz, ArB-H5), 6.903 (1H, d, J = 7.8 Hz, ArA-H5), 4.140 (2H, t, J = 6 Hz,
-OCH2C-), 3.915 (3H, s, ArA-OCH3), 3.904 (3H, s, ArB-OCH3), 3.875 (3H, s, ArB-OCH3), 3.839 (4H, s,
morpholine-H3,5), 2.931 (4H, t, J = 5.4 Hz, cyclohexanone-H3,5), 2.381 (2H, t, J = 6 Hz, -OCCCH2-), 3.727 (4H, s,
morpholine-H2,6), 1.913-1.928 (2H, m, -OCCH2C-), 1.808-1.828 (2H, m, cyclohexanone-H4). ESI-MS m/z: 508.4
(M+1)+, calcd for C30H37NO6: 507.6.
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4.1.6.14
one(36)
(2E,6E)-2-[3-methoxy-4-(3-morpholinopropoxy)benzylidene]-6-(2-methoxybenzylidene)cyclohexan
1
Yellow oil. H-NMR (600 MHz, DMSO-d6): δ(ppm) 7.790 (1H, s, ArB-CH=), 7.701 (1H, d, J = 7.2 Hz, ArB-H6),
7.597 (1H, s, ArA-CH=), 7.346-7.370 (1H, m, ArB-H4), 6.983-7.135 (5H, m, ArA-H2,5,6, ArB-H3,5), 4.034 (2H, t, J =
6.6 Hz, -OCH2-), 3.826 (3H, s, ArB-OCH3), 3.798 (3H, s, ArA-OCH3), 3.561 (4H, s, morpholine-H3,5), 2.887 (4H, t,
J = 6.6 Hz, cyclohexanone-H3,5), 2.407 (2H, t, J =6.6Hz, -OCCCH2-), 2.346 (4H, s, morpholine-H2,6), 1.858-1.889
(2H, m, -OCCH2C-), 1.651-1.680 (2H, m, cyclohexanone-H4). ESI-MS m/z: 478.4 (M+1)+, calcd for C29H35NO5:
477.6.
4.1.6.15 (2E,5E)-2-[3-methoxy-4-(3-morpholinopropoxy)benzylidene]-5-[2-(trifluoromethyl)benzylidene]cyclo
pentanone(37)
1
Yellow oil. H-NMR (600 MHz, CDCl3): δ(ppm) 7.808 (1H, s, ArB-CH=C-), 7.714 (1H, d, J = 7.8 Hz, ArB-H3) ,
7.327-7.669 (5H, m, ArB-CH=C-, ArA-H2,6, ArB-H5,6), 7.043-7.107 (1H, m, ArB-H4), 6.938 (1H, d, J = 8.4 Hz,
ArA-H5), 4.129 (2H, d, J = 6.6 Hz, -OCH2C-), 3.883 (3H, s, -OCH3), 3.847 (4H, s, morpholine-H3,5), 3.025 (2H, s,
cyclopentanone-H3), 2.952 (2H, s, cyclopentanone-H4), 2.557 (2H, t, J = 6.6 Hz, -OCCCH2-), 2.486 (4H, s,
morpholine-H2,6), 1.965-2.002 (2H, m, -OCCH2C-). ESI-MS m/z: 502.1 (M+1)+, calcd for C28H30F3NO4: 501.5.
4.2 Cell Lines and Culture
Two human gastric carcinoma cell lines SGC-7901 and MGC80-3, human colorectal cancer cell line SW-620,
mouse colorectal cancer cell line CT-26, and the human gastric mucosa epithelial cell line GES-1 were obtained
from the cell bank of the Chinese Academy of Sciences and grown in RPMI-1640 medium (Gibico) supplemented
with 10% (v/v) heat-inactivated fetal bovine serum (Gibico) and antibiotics (100 U/mL penicillin and 100µg/mL
streptomycin). Cells were maintained in a humidified cell incubator at 37°C with 5% CO2.
4.3 Reagents
Antibodies against Bcl-2(sc-492), Pro-caspase 3 (sc-7148), cleaved PARP (sc-56196), GAPDH (sc-25778)
and KI67 (sc-15402) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); Antibody against
Cleaved-caspase 3 (Asp175) was purchased from Cell Signaling Technology
4.4 Cell Growth Inhibition Assays (MTS assay)
Cell lines were seeded in 96-well plates in triplicate at a concentration of 4000 cells / well and allowed to
attach overnight. Cells were then exposure to various concentrations of test compounds in triplicate for 72 h under
5% CO2 at 37°C. MTS ((Promega, San Luis Obispo, CA)) was used according to manufacturer instructions.
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Briefly, the MTS solution was added and the culture was incubated for an additional 4 h and absorbance was
measured by using the spectraMax M2 microplate reader (MolecularDevices, Sunnyvale, CA) at 490 nm. The
results were also expressed as IC50 (the compound concentration required for 50% growth inhibition of tumor
cells), which was calculated by the software Origin75. The mean ± SEM IC50 was determined from the results of
three independent tests.
4.5 Cell Apoptosis Assay
Apoptosis was determined using an apoptosis detection Kit (BD Biosciences, USA). Briefly, SGC-7901 cells
were seeded into 6-well plates and allowed grown to 80% confluency in complete medium, then cells treated with
different concentrations of test compounds for 30h to evaluate the effects of test compounds on apoptosis. Cells
were collected, washed twice in ice-cold phosphate-buffered saline (PBS), and then resuspended in binding buffer
according to the instructions of the apoptosis Kit. The treated cells (as described above) were simultaneously
incubated with fluorescein-labeled Annexin V and PI. Annexin V-binding buffer was then added to the mixture
before fluorescence was measured on a FACSCalibur (BD Biosciences; Baltimore, MD, USA). Data were
analyzed using Flowjo software.
4.6 Western Blot Analysis
SGC-7901 cells were treated with test compounds for 24 hours, then harvested, washed twice with PBS, lysed
on ice for 30min in 80ꢀL of lysis buffer to extract total protein, and then centrifuged at 12,000 ×rpm for 15 min.
The supernatants were collected from the lysates and the protein concentrations were determined. Aliquots of the
lysates (60ꢀg of protein) were boiled for 10 min and electrophoresed in 10% sodium dodecyl
sulfate-polyacrylamide gel. Separated protein bands were transferred into polyvinylidene fluoride (PVDF)
membranes and the membranes were blocked in 5% skimmed milk. The primary antibodies against cleaved-PARP,
pro-caspase3 and Bcl-2 were diluted according to the manufacturer's instructions and incubated overnight at 4˚C.
Horseradish peroxidase-linked secondary antibodies (Santa cruze) were then added at a dilution ratio of 1:300, and
incubated at room temperature for 1 h. Protein bands were detected using an EZ-ECL Chemiluminescence
Detection Kit (Biological Industries).
4.7 Antitumor effect of compound 28 in xenograft model
Female nude mice were obtained from Animal Center of Wenzhou Medical University (Wenzhou, China) and
protocols involving use of animals were approved by the Wenzhou Medical University Animal Policy and Welfare
Committee. Eight-week-old female mice were injected subcutaneously with 10 million SGC7901 cells. To
determine whether compound 28 inhibits tumor formation after engraftment of cells, treatment was initiated when
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tumors reached about 40 mm3, and animals were randomized to receive 0.5% CMCNA alone, compound 28 (50
mg/kg/day, orally), compound 28 (100 mg/kg/day, orally) and curcumin (50mg/kg/day, orally) for 23 consecutive
days. Mice were observed daily and tumor size was measured every 2 days using calipers. Volume was calculated
2
using the formula: π/6 × (large diameter) × (small diameter) . Tumor samples and organs were collected when
mice were sacrificed. Each sample was cut in halves; one half was preserved in 4% paraformaldehyde and one half
was flash-frozen in liquid nitrogen and stored at −80°C until further use.
4.8 Immunohistochemistry
5ꢀm sections were deparaffinized with xylene and rehydrated through graded alcohols into water. Then,
antigen retrieval was carried out for 10 minutes in sodium citrate buffer. Tissues were placed in 3% H2O2 for 10
minutes to increased permeability, followed by incubation in 1% bovine serum albumin in PBS for 10 minutes,
washing twice in PBS. Slides were then incubated with the primary antibodes overnight at 4°C, monoclonal rabbit
anti-Ki67 (Santa Cruz, sc-15402) at a 1:50 dilution, cleaved-caspase 3 (Cell Signaling Technology, Asp175) at a
1:100 dilution. Following overnight incubation, slides were washed and incubated with horseradish
peroxidase-conjugated secondary antibody for 2h at 37°C with 1:100 dilution. The tissue sections were then
washed and stained with DAB substrate-chromogen and then counterstained with hematoxylin, dehydrated,
cleared, and mounted with coverslips. Electronic images were captured using microscope.
4.9 Statistical Analysis
Data were presented as the mean ± SEM. Differences between groups were analyzed using the Student’s t-test
for continuous variables. Statistical analysis was performed using Graphpad prism (version 5) and significance was
established at p < 0.05.
Acknowledgements
Financial support was provided by the Natural Science Funding of Zhejiang Province (LY13H160022), National
Natural Science Foundation of China (81302642, 81402783, and 81473242), Zhejiang Key Health Science and
Technology Project (WKJ2013-2-021).
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Figure legends
Table 1. Inhibition of cell proliferation by compounds in four cancer cell lines and GES-1 gastric mucosa
epithelial cell line. The IC50 values were determined as described in “Materials and methods”, Data were obtained
from three independent experiments.
Figure 1. The anti-cancer structural-activity relationship of mono-carbonyl analogues of curcumin and the
chemical structures of anticancer drugs that including a long-chain alkoxyl group.
Figure 2. General synthesis and chemical structures of long-chain alkoxylated mono-carbonyl analogues of
curcumin (1-22).
Figure 3. General synthesis and chemical structures of long-chain alkoxylated mono-carbonyl analogues of
curcumin (23-37).
Figure 4. Apoptosis induced by compounds 5, 28, 29 treatment in SGC7901 gastric cancer. UR (up right): necrotic
cells and late apoptotic cells labeled with PI and Annexin V-FITC. LL (lower left): fully viable cells. LR (lower
right): early apoptotic cells labeled with Annexin V-FITC but not with PI. Cells were incubated with the indicated
doses (2.5, 5 or 10 ꢀM) of compounds 5, 28, 29 for 30h (DMSO as negative control, cur as positive control), and
then harvested and analyzed by flow cytometry. Representative data from one experiment are shown, the average
of three separate experiments are shown, significant difference from control was indicated as * character (*p <
0.05).
Figure 5. The expression of cleaved-PARP, Caspase3 and total Bcl-2 after exposure to test compounds in
SGC7901 cell. (A) SGC7901were treated for 24 h with compounds 5, 28, 29 at indicated doses (2.5, 5 or 10 ꢀM)
and whole cell extracts were prepared. Protein expression was then analyzed by western blot. (B) cleaved-PARP;
(C) Caspase3; (D) Bcl-2. Data are representative of three individual experiments. (* represents p < 0.05)
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Figure 6. Schedule dependence of in vivo antitumor activity of compound 28 against the SGC7901 xenograft
model. (A) Established (~ 40 mm3) SGC7901 tumors were administered compound 28 on a once (QD x 23, orally)
dosing schedule. For comparison, vehicle group of 0.5% CMCNA, positive control group of cur (50 mg/kg, PO)
were included. %ꢁT/ꢁC was determined. Data plotted as average tumor volume ± SEM. Compound 28 causes
tumor regressions when dosed at 50 mg/kg/ QD and 100 mg/kg QD schedule. (B) Tumors were weighed after
sacrificing the mice on the final day of the study. Compound 28 dose-dependently diminished the tumor weight. (C)
Body weights were monitored for the duration of the studies. * indicates P < 0.05.
Figure 7. Subcutaneous SGC7901 xenografts, treated with a 50 mg/kg or 100 mg/kg dose of compound 28 were
examined KI67 and Cleaved-caspase3 status by immunohistochemistry. Top panel, expression of KI67 in tumor
tissue. Representative images were taken from scans at 40x magnification. Bottom panel, expression of
Cleaved-caspase3 in tumor tissue. Representative images were taken from scans at 20x magnification.
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Table 1
IC50 (μM)
SW620
Comp.
SGC7901
MGC803
CT26
GES-1
1
2
4.96±1.3
10.99±0.92 10.24±0.19
2.06±0.95
1.98±0.08 2.69±0.09 3.02±0.03
5.64±0.9 10.52±0.44 10.04±0.15
4
5
6
7
1.88±0.09
1.7±0.06
3.38±0.05
2.32±0.09
1.94±0.4
1.72±0.11
2.21±0.09
2.92±0.42
1.04±0.06
0.99±0.02 2.06±0.03 3.45±0.08
2.79±0.07 3.59±0.2 2.87±0.03
2.49±0.15 2.42±0.26 6.35±0.59
2.01±0.3
3.27±0.05
9
11.89±1.53 15.48±0.41 4.65±2.42 12.82±0.25 14.08±1.05
12
16
17
18
19
25
27
28
29
30
31
32
35
6.26±2.24
0.89±0.31
1±0.1
7.72±0.75
2.46±1.28
2.28±0.91
0.68±0.08
6.26±0.62 9.85±0.33 3.56±0.28
0.95±0.53 0.36±0.03 7.09±1.71
1.49±0.32 0.39±0.05
>30
0.44±0.03
5.08±1.31
5.99±1.28
7.67±1.25
1.91±0.06
1.84±0.04
2.84±0.11
4.52±1.45
2.77±0.06
2.87±0.14
0.39±0.03 0.18±0.04 0.67±0.18
7.31±0.03 4.47±0.003 3.86±0.38 7.03±0.003
7.14±0.22
8.46±1.16
2.18±0.41
2.49±0.25
6.69±1.05
5.52±1.34
2.45±0.44
8.61±0.5
2.8±0.12 3.13±0.09 7.81±0.26
3.38±0.19 7.01±0.36 8.06±0.33
1.22±0.05 2.56±0.05 2.62±0.19
1.02±0.03 2.38±0.53 2.77±0.01
3.53±0.22 6.39±0.64 4.11±0.25
4.43±0.16 4.51±1.12 4.04±0.18
1.62±0.16 3.26±0.62 3.03±0.02
3.81±0.09 2.99±0.62 8.17±0.43
CUR 24.41±4.76 31.39±13.09 12.52±1.31 7.7±1.05
27.89±2.3

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Figure 1
N
N
N
O
O
CN
O
Cl
Cl

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Figure 2

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Figure 3