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Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Synthesis, anti-tumor activity, and structure–activity relationships of curcumol derivatives ab
b
b
b
Ping Guo , Yue-Wu Wang , Bi-Xia Weng , Xiao-Kun Li , Shu-Lin a
Yang & Fa-Qing Ye
b
a
Institute of Chemical Technology, Nanjing University of Science and Technology, Nanjing, 210094, China b
School of Pharmacy, Wenzhou Medical College, Wenzhou, 325035, China Published online: 25 Nov 2013.
To cite this article: Ping Guo, Yue-Wu Wang, Bi-Xia Weng, Xiao-Kun Li, Shu-Lin Yang & Fa-Qing Ye (2014) Synthesis, anti-tumor activity, and structure–activity relationships of curcumol derivatives, Journal of Asian Natural Products Research, 16:1, 53-58, DOI: 10.1080/10286020.2013.857660 To link to this article: http://dx.doi.org/10.1080/10286020.2013.857660
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Journal of Asian Natural Products Research, 2014 Vol. 16, No. 1, 53–58, http://dx.doi.org/10.1080/10286020.2013.857660
Synthesis, anti-tumor activity, and structure – activity relationships of curcumol derivatives Ping Guoab, Yue-Wu Wangb, Bi-Xia Wengb, Xiao-Kun Lib, Shu-Lin Yanga* and Fa-Qing Yeb* a
Institute of Chemical Technology, Nanjing University of Science and Technology, Nanjing 210094, China; bSchool of Pharmacy, Wenzhou Medical College, Wenzhou 325035, China
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(Received 10 April 2013; final version received 17 October 2013) Using curcumol that was extracted from the volatile oil of Rhizoma Curcumae as the raw material, its derivatives were synthesized and purified. The structures of these compounds were confirmed by 1H, 13C NMR, and mass spectral data. The test compounds were evaluated for their in vitro anti-tumor activity against gastric cancer cell lines SGC-7901 and lung carcinoma cell line H460 by methyl thiazolyl tetrazolium chromatometry. Distinct structure – activity relationships of these curcumol derivatives were also revealed for inhibiting cell proliferation. Presence of electron-withdrawing groups or amino could increase the activity significantly, whereas esterification of 8hydroxy diminished the anti-tumor activity. Many of the tested candidates exhibited higher inhibition efficiency than curcumol, suggesting that structural modifications could enhance its activity effectively. Keywords: curcumol; synthesis; derivatives; anti-tumor activity; structure – activity relationships
1.
Introduction
Rhizoma Curcumae (rhizome of Curcuma Zedoaria; Ezhu in Chinese), a common Traditional Chinese Medicine, has been applied for the prevention and treatment of skin and hepatic conditions and of ulcers and digestive disorders, and has also been used in the treatment of intestinal parasites and as a remedy for poisoning, snakebites, and various other complaints [1,2]. The essential oil had been extracted from Rhizoma Curcumae and used to treat cancer in China. Curcumol, an important active component of the oil, is commonly used as a quality control marker [3]. According to the relational grade and relational sequence between fingerprint of essential oil and inhibiting proliferation rate, the contribution to inhibit proliferation of nasopharyngeal carcinoma cell was curcumol . curdione . b-elemene . germacrone .
curzerenone [4]. And curcumol could be transformed by a quantitative isomerization from curdione, another major active ingredient of the essential oil [5]. Subsequently, curcumol became the key biological compound in Rhizoma Curcumae, and its bioactivity had recently attracted more attention. Curcumol, 5 – 7-membered guaianetype sesquiterpenes (Figure 1), was isolated from the rhizome of zedoary (Curcuma zedoaria Roscoe) firstly by Hiroshi in 1965 [6]. It has been reported to possess anti-tumor activity against a broad range of human cancer cells with low cytotoxicity. Curcumol can induce apoptosis and inhibit the proliferation of many kinds of tumor cells, such as gastric cancer, nasopharyngeal carcinoma, cervical cancer, and lung carcinoma cells [7 – 9]. But the significant inhibition of prolifer-
*Corresponding authors. Email: [email protected]; [email protected] q 2013 Taylor & Francis
54
P. Guo et al. derivative of curcumol, had been synthesized, and its structure was confirmed by single-crystal X-ray diffraction in our previous works [10]. The compound that had been isolated from the microbial oxidation products of curcumol [11] could be obtained easily through an improved chemical method. To study the structure –activity relationship, its analogs had been synthesized (Scheme 1). These derivatives of curcumol were assayed for their anti-tumor activity against gastric cancer cell lines SGC7901 and lung carcinoma cell line H460 (Table 1). The weak activity of compound 1 indicated that the special cyclic structure is important to combine with its biological target. By contrasting the activity of monoester and diester, the free 8-hydroxy is necessary to retain its anti-tumor activity. In the presence of electronwithdrawing phenyl group or amino group in the chain, many compounds showed outstanding efficiency to inhibit cell proliferation. The abnormal activity of compound 5 is possible due to its large stereospecific blockade. To improve the anti-tumor activity further, it is necessary to maintain the same cyclic structure and free 8-hydroxyl and meanwhile to import nitrogen atom into the structure (Figure 2).
H
O
OH
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Figure 1. The chemical structure of curcumol.
ation cannot be observed currently when the cells were dealed with curcumol under the concentration of 50 mg/ml. Obviously, it lacks enough efficiency to become a clinical drug. To improve its anti-tumor activity, a series of derivatives were synthesized starting from curcumol that was extracted from the volatile oil of Rhizoma Curcumae.
2.
Results and discussion
Rhizoma Curcumae oil (commercially available) was distilled in vacuum to remove volatile component with low boiling point. The residue was extracted repeatedly by petroleum ether. After removal of the extracted liquid, the viscous materials were crystallized in ethanol. Curcumol with 98% purity can be separated into 10–15% yield. In the molecular structure of curcumol, the number of H-bond donors and acceptors is little. The combination reaction between small molecular and corresponding biological target is not strong enough. It is unfavorable to exhibit the activity of curcumol. So, it is necessary to introduce more active groups such as hydroxyl and amino into the chemical structure of curcumol. Compound 1, a
3. Experimental 3.1 General experimental procedures Uncorrected melting points (m.p.) were determined using an X-4 (Gongyi, Henan, China) micro-melting point apparatus. 1H NMR and 13C NMR spectra were recorded OCOR
OH
H
1)m-CPBA O
Curcumol
OH
H
RCOCl O
2)NaOH 1
Scheme 1. Synthetic route of curcumol derivatives.
OH
or RCOOH
H O
OCOR
Journal of Asian Natural Products Research
solution to remove the residue m-CPBA. The organic phase was passed through anhydrous sodium sulfate to remove traces of water, and the solvent was evaporated under vacuum to obtain the mixture of epoxy derivatives. The mixture and solid NaOH were added into 20 ml ethanol, and the contents were refluxed for 2 h. To dump the solution into 100 ml water, product 1 was crystallized directly in 50% yield from this water. The synthetic route of product 2 was described in Ref. [10].
Table 1. The anti-tumor activity of curcumol derivatives.
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Compounds 1 2 3 4 5 6 7 8 Curcumol
SGC-7901 (mg/ml)
H460 (mg/ml)
175.3 168.7 . 200 . 200 34.7 37.7 24.0 120.8 72.6
196.4 173.6 . 200 . 200 47.8 40.5 17.1 150.3 78.5
3.3 General procedure for the preparation of diester derivatives
on a Bruker Avance-3600 (Bruker Technologies, Bremen, Germany) Spectrometer (600 MHz). Samples were dissolved in CDCl3, while tetramethylsilane (TMS) was used as an internal standard. Chemical shifts were recorded in (ppm) values relative to TMS, and J values were expressed in Hertz. MS were recorded using an Agilent-1100 LC – MS Spectrometer (Agilent Technologies, Waldbronn, Germany). The relative chemical reagents were purchased from Aladdin-reagent (Shanghai, China).
3.2
To a solution of 1 in dichloromethane, 2– 3 drops of triethylamine were added. The respective acid chloride dissolved in dichloromethane was added dropwise. The reaction mixture was stirred for 3 h with a magnetic stirrer, and the progress of the reaction was monitored by TLC. The products were purified by silica gel column chromatography using ethyl acetate and petroleum ether as the eluting solvent. The diester derivatives were obtained with 60 –80% yield.
Preparation of derivative 1
3.3.1
R1
14 2
15
10
1
9
O
3
Compound 3
A colorless oil, 1H NMR (CDCl3, 600 MHz) d: 0.82 (d, 3H, J ¼ 6.6 Hz, H-13), 0.85 (d, 3H, J ¼ 6.6 Hz, H-12), 0.93 (d, 3H, J ¼ 6.6 Hz, H-15), 1.13–1.25 (m, 1H, H6a), 1.41–1.46 (m, 1H, H-11), 1.50–1.55 (m, 1H, H-2a), 1.54–1.58 (m, 1H, H-3a), 1.74–1.77 (m, 1H, H-7), 1.76–1.80 (m, 1H, H-4), 1.79–1.84 (m, 1H, H-3b), 1.90–1.94
Curcumol (2.36 g) dissolved in 30 ml dichloromethane reacted with m-CPBA (3-chloroperbenzoic acid, 2.58 g) at room temperature. The progress of the reaction was monitored by thin-layer chromatography (TLC). After completion of the reaction, the reaction liquid was washed by sodium sulfite and sodium hydroxide
H
8
45 6
55
7
R2 13 11
Compound 1: R1=R2=OH; 2: R1=I, R2=OH; 3: R1=R2=CH3COO4: R1=R2=4-methyl-benzoyloxy; 5: R1=4-nitro-benzoyloxy, R2=OH 6: R1=4-chloro-benzoyloxy, R2=OH; 7: R1=Boc-Glycine, R2=OH 8: R1=Boc-Phenylalanine, R2=OH
Figure 2. Structures of the tested curcumol derivatives.
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P. Guo et al.
(m, 1H, H-2b), 1.98 (s, 3H, ZCOCH3), 2.00 (s, 3H, ZCOCH3), 2.02–2.06 (m, 1H, H-1), 2.09 (m, 1H, H-6b), 4.41 (d, 1H, J ¼ 13.2 Hz, H-14), 4.47 (d, 1H, J ¼ 13.2 Hz, H-14), 5.81 (s, 1H, H-9); ESI-MS: m/z 359.1 [M þ Na]þ (100%). 13 C NMR (CDCl3, 150.9 MHz) d: 11.5 (C-15), 20.8 (ZOCOCH3), 21.1 (C-12), 22.0 (ZOCOCH3), 22.4 (C-13), 27.4 (C-2), 30.3 (C-11), 31.0 (C-3), 34.9 (C-6), 40.1 (C-4), 49.5 (C-1), 57.3 (C-7), 65.6 (C-14), 88.7 (C-5), 105.2 (C-8), 125.9 (C-9), 136.1 (C-10), 168.3 (ZOCO), 170.6 (ZOCO). 3.3.2
Compound 4
diimide was added. After stirring the mixture for 30 min, an equimolecular quantity of compound 1 was added. The products were separated from the mixture of reactions by silica gel column chromatography with 40– 60% yield. In the HMBC spectra of these compounds, significant correlations from proton H-14 to the involved carbonyl carbon could be observed. Obvious downfield shift of H-14 indicated that C-14 was linked with the ester group. These findings confirmed that esterification occurred only at the site of 14-OH. 3.4.1
1
A colorless oil, H NMR (CDCl3 , 600 MHz) d: 0.95 (d, 3H, J ¼ 6.6 Hz, H13), 1.02 (d, 3H, J ¼ 9.0 Hz, H-12), 1.03 (d, 3H, J ¼ 6.6 Hz, H-15), 1.24 –1.30 (m, 1H, H-6a), 1.59– 1.64 (m, 1H, H-11), 1.64– 1.69 (m, 1H, H-2a), 1.67 –1.72 (m, 1H, H3a), 1.80 – 1.84 (m, 1H, H-7), 1.92 – 1.97 (m, 1H, H-4), 1.90– 1.94 (m, 1H, H-3b), 1.88 –1.92 (m, 1H, H-2b), 2.10– 2.15 (m, 1H, H-1), 2.23– 2.28 (m, 1H, H-6b), 2.40 (s, 3H, ZPhZCH 3), 2.42 (s, 3H, ZPhZCH3), 4.80 (d, 1H, J ¼ 13.2 Hz, H14), 4.81 (d, 1H, J ¼ 13.2 Hz, H-14), 6.08 (s, 1H, H-9), 7.22 –7.27 (m, 4H, PhZH), 7.93 –8.01 (m, 4H, PhZH); ESI-MS: m/z 511.2 [M þ Na]þ (100%). 13 C NMR (CDCl3, 150.9 MHz) d: 11.8 (C-15), 21.8 (ZPhZCH3), 21.8 (ZPhZCH3), 21.9 (C-12), 22.7 (C-13), 27.9 (C-2), 30.6 (C-11), 31.2 (C-3), 35.2 (C-6), 40.4 (C-4), 50.0 (C-1), 58.4 (C-7), 66.4 (C-14), 89.2 (C-5), 105.7 (C-8), 126.5 (Ph-10 ), 126.8 (Ph-100 ), 129.3 (Ph-30 ,50 ), 129.6 (Ph-300 ,500 ), 130.1 (Ph-20 ,60 ), 130.4 (Ph-200 ,600 ), 136.6 (C-9), 143.8 (Ph-40 ), 143.9 (Ph-400 ), 144.7 (C-10), 164.1 (ZOCO), 166.5 (ZOCO). 3.4 General procedure for the preparation of monoester derivatives When the acid was dissolved in dichloromethane, excess of 1,3-dicyclohexylcarbo-
Compound 5
A yellow solid, m.p. 113–1168C; 1H NMR (CDCl3, 600 MHz) d: 0.85 (d, 3H, J ¼ 6.6 Hz, H-13), 0.96 (d, 3H, J ¼ 9.0 Hz, H-15), 0.98 (d, 3H, J ¼ 6.6 Hz, H-12), 1.22– 1.25 (m, 1H, H-6a), 1.44–1.46 (m, 1H, H11), 1.56–1.57 (m, 1H, H-2a), 1.59–1.60 (m, 1H, H-3a), 1.67–1.69 (m, 1H, H-7), 1.82–1.83 (m, 1H, H-4), 1.87–1.89 (m, 1H, H-3b), 1.89–1.91 (m, 1H, H-2b), 2.02–2.18 (m, 1H, H-1), 2.21 (dd, 1H, J ¼ 13.2 Hz, 10.8 Hz, H-6b), 4.75 (d, 1H, J ¼ 13.2 Hz, H14), 4.82 (d, 1H, J ¼ 13.2 Hz, H-14), 5.93 (s, 1H, H-9), 8.17 (d, 2H, J ¼ 9.0 Hz, PhZH), 8.26 (d, 2H, J ¼ 9.0 Hz, PhZH). 13 C NMR (CDCl3, 150.9 MHz) d: 11.7 (C-15), 21.8 (C-12), 22.6 (C-13), 27.6 (C-2), 30.6 (C-11), 31.2 (C-3), 36.4 (C-6), 40.2 (C4), 50.0 (C-1), 59.4 (C-7), 67.0 (C-14), 87.3 (C-5), 103.3 (C-8), 123.8 (Ph-30 ,50 ), 128.0 (C-9), 130.9 (Ph-20 ,60 ), 135.5 (Ph-10 ), 138.1 (C-10), 150.6 (Ph-40 ), 164.3 (CvO); ESIMS: m/z 402.3 [M þ H]þ (100%). 3.4.2
Compound 6
A white solid, m.p. 105 –1068C, 1H NMR (CDCl3, 600 MHz) d: 0.87 (d, 3H, J ¼ 6.6 Hz, H-13), 0.98 (d, 3H, J ¼ 6.6 Hz, H-12), 1.01 (d, 3H, J ¼ 6.6 Hz, H-15), 1.24 (m, 1H, H-6a), 1.48 – 1.50 (m, 1H, H11), 1.55– 1.59 (m, 1H, H-2a), 1.59– 1.64 (m, 1H, H-3a), 1.65 –1.68 (m, 1H, H-7), 1.82 –1.84 (m, 1H, H-4), 1.84 – 1.89 (m,
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Journal of Asian Natural Products Research 1H, H-2b), 1.90– 1.92 (m, 1H, H-3b), 2.03 –2.05 (m, 1H, H-1), 2.19 –2.23 (m, 1H, H-6b), 4.71 (d, 1H, J ¼ 13.2 Hz, H14), 4.78 (d, 1H, J ¼ 13.2 Hz, H-14), 5.93 (s, 1H, H-9), 7.40 (d, 2H, J ¼ 8.4 Hz, Ph-H), 7.95 (d, 2H, J ¼ 8.4 Hz, PhZH). 13 C NMR (CDCl3, 150.9 MHz) d: 11.8 (C-15), 21.5 (C-12), 22.7 (C-13), 27.7 (C2), 30.9 (C-11), 31.3 (C-3), 36.5 (C-6), 40.3 (C-4), 50.1 (C-1), 59.6 (C-7), 66.4 (C-14), 87.4 (C-5), 103.4 (C-8), 127.3 (C-9), 128.0 (Ph-10 ), 128.9 (Ph-30 ,50 ), 131.1 (Ph-20 ,60 ), 138.8 (C-10), 165.4 (CvO), 139.7 (Ph-40 ); ESI-MS: m/z 391.4 [M þ H]þ (100%). 3.4.3 Compound 7 A white solid, m.p. 88 –908C, 1H NMR (CDCl 3, 600 MHz) d: 0.84 (d, 3H, J ¼ 6.6 Hz, H-13), 0.95 (d, 3H, J ¼ 6.6 Hz, H-12), 0.98 (d, 3H, J ¼ 6.6 Hz, H-15), 1.18 –1.22 (m, 1H, H-6a), 139– 1.42 (m, 1H, H-11), 1.42 (d, 9H, J ¼ 1.2 Hz, ZC(CH3)3), 1.49– 1.51 (m, 1H, H-2a), 1.51– 1.55 (m, 1H, H-3a), 1.61 –1.66 (m, 1H, H-7), 1.78 –1.82 (m, 1H, H-4), 1.82– 1.86 (m, 1H, H-2b), 1.85– 1.89 (m, 1H, H-3b), 1.94 –1.98 (m, 1H, H1), 2.15 – 2.19 (m, 1H, H-6b), 3.90 (d, 2H, J ¼ 4.8 Hz, ZCH2NH), 4.51 (d, 1H, J ¼ 13.2 Hz, H-14), 4.58 (d, 1H, J ¼ 13.2 Hz, H-14), 5.82 (s, 1H, H-9), 3.60 (br, 1H, ZOH), 5.12 (br, 1H, ZNH). 13 C NMR (CDCl3, 150.9 MHz) d: 11.7 (C-15), 21.5 (C-12), 22.7 (C-13), 27.5 (C-2), 29.8 (Boc-Gly), 30.8 (C-11), 31.2 (C-3), 36.4 (C-6), 40.3 (C-4), 42.5 (Boc-Gly), 49.7 (C-1), 59.4 (C-7), 66.4 (C-14), 80.1 (Boc-Gly), 87.2 (C-5), 103.3 (C-8), 127.8 (C-9), 138.3 (C-10), 155.8 (Boc-Gly), 170.3 (Boc-Gly); ESI-MS: m/z 410.2 [M þ H]þ (100%). 3.4.4
Compound 8
A white solid, m.p. 74 –778C, 1H NMR (CDCl 3, 600 MHz) d: 0.86 (d, 3H, J ¼ 6.6 Hz, H-13), 0.98 (d, 3H, J ¼ 6.6 Hz, H-12), 1.00 (d, 3H, J ¼ 6.6 Hz, H-15), 1.16 – 1.21 (m, 1H, H-6b), 1.38 –1.41 (m, 1H, H-11), 1.41 (d, 9H,
57
J ¼ 1.2 Hz, ZC(CH 3) 3), 1.47 – 1.51 (m, 1H, H-2a), 1.53 – 1.58 (m, 1H, H3a), 1.62– 1.67 (m, 1H, H-7), 1.78– 1.82 (m, 1H, H-4), 1.77 –1.81 (m, 1H, H-2b), 1.84 –1.88 (m, 1H, H-3b), 1.88 –1.93 (m, 1H, H-1), 2.15 –2.20 (m, 1H, H-6a), 4.43 (d, 1H, J ¼ 13.2 Hz, H-14), 4.57 (d, 1H, J ¼ 13.2 Hz, H-14), 5.79 (s, 1H, H-9), 3.00 –3.12 (m, 2H, ZCH2Ph), 4.52– 4.63 (m, 1H, ZCHNH), 7.12 – 7.28 (m, 5H, PhZH), 3.30 (br, 1H, ZOH), 5.01 (br, 1H, ZNH). 13 C NMR (CDCl3, 150.9 MHz) d: 11.6 (C-15), 21.2 (C-12), 22.1 (C-13), 27.5 (C-2), 28.4 (Boc-Phe), 30.8 (C-11), 31.2 (C-3), 36.4 (C-6), 38.5 (Boc-Phe), 40.3 (C-4), 49.6 (C1), 54.6 (Boc-Phe), 59.5 (C-7), 66.5 (C-14), 80.0 (Boc-Phe), 87.1 (C-5), 103.3 (C-8), 125.5 (Boc-Phe), 127.1 (Boc-Phe), 127.8 (C9), 128.5 (Boc-Phe), 128.6 (Boc-Phe), 129.3 (Boc-Phe), 136.1 (Boc-Phe), 138.3 (C-10), 155.2 (Boc-Phe), 171.8 (Boc-Phe); ESI-MS: m/z 499.9 [M þ H]þ (100%).
3.5
Bioassay
To conduct the methyl thiazolyl tetrazolium (MTT) assay, the cells (3000 per well) were seeded in wells of a 96-well plate in 100 ml of complete media, and incubated for 3 days at 378C. Then 20 ml of MTT dissolved at 5 mg/ml in sterile phosphatebuffered saline was added to the cells. Four hours later, the media was removed, the metabolized MTT was suspended in dimethyl sulfoxide, and the absorbance was measured at 490 nm. The experiment was conducted in triplicates, and the statistical analysis (simple linear regression analysis initially and then unpaired Student’s test that revealed significant differences between two sample means) was carried out to obtain the final values.
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Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Synthesis, anti-tumor activity, and structure–activity relationships of curcumol derivatives ab
b
b
b
Ping Guo , Yue-Wu Wang , Bi-Xia Weng , Xiao-Kun Li , Shu-Lin a
Yang & Fa-Qing Ye
b
a
Institute of Chemical Technology, Nanjing University of Science and Technology, Nanjing, 210094, China b
School of Pharmacy, Wenzhou Medical College, Wenzhou, 325035, China Published online: 25 Nov 2013.
To cite this article: Ping Guo, Yue-Wu Wang, Bi-Xia Weng, Xiao-Kun Li, Shu-Lin Yang & Fa-Qing Ye (2014) Synthesis, anti-tumor activity, and structure–activity relationships of curcumol derivatives, Journal of Asian Natural Products Research, 16:1, 53-58, DOI: 10.1080/10286020.2013.857660 To link to this article: http://dx.doi.org/10.1080/10286020.2013.857660
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Journal of Asian Natural Products Research, 2014 Vol. 16, No. 1, 53–58, http://dx.doi.org/10.1080/10286020.2013.857660
Synthesis, anti-tumor activity, and structure – activity relationships of curcumol derivatives Ping Guoab, Yue-Wu Wangb, Bi-Xia Wengb, Xiao-Kun Lib, Shu-Lin Yanga* and Fa-Qing Yeb* a
Institute of Chemical Technology, Nanjing University of Science and Technology, Nanjing 210094, China; bSchool of Pharmacy, Wenzhou Medical College, Wenzhou 325035, China
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(Received 10 April 2013; final version received 17 October 2013) Using curcumol that was extracted from the volatile oil of Rhizoma Curcumae as the raw material, its derivatives were synthesized and purified. The structures of these compounds were confirmed by 1H, 13C NMR, and mass spectral data. The test compounds were evaluated for their in vitro anti-tumor activity against gastric cancer cell lines SGC-7901 and lung carcinoma cell line H460 by methyl thiazolyl tetrazolium chromatometry. Distinct structure – activity relationships of these curcumol derivatives were also revealed for inhibiting cell proliferation. Presence of electron-withdrawing groups or amino could increase the activity significantly, whereas esterification of 8hydroxy diminished the anti-tumor activity. Many of the tested candidates exhibited higher inhibition efficiency than curcumol, suggesting that structural modifications could enhance its activity effectively. Keywords: curcumol; synthesis; derivatives; anti-tumor activity; structure – activity relationships
1.
Introduction
Rhizoma Curcumae (rhizome of Curcuma Zedoaria; Ezhu in Chinese), a common Traditional Chinese Medicine, has been applied for the prevention and treatment of skin and hepatic conditions and of ulcers and digestive disorders, and has also been used in the treatment of intestinal parasites and as a remedy for poisoning, snakebites, and various other complaints [1,2]. The essential oil had been extracted from Rhizoma Curcumae and used to treat cancer in China. Curcumol, an important active component of the oil, is commonly used as a quality control marker [3]. According to the relational grade and relational sequence between fingerprint of essential oil and inhibiting proliferation rate, the contribution to inhibit proliferation of nasopharyngeal carcinoma cell was curcumol . curdione . b-elemene . germacrone .
curzerenone [4]. And curcumol could be transformed by a quantitative isomerization from curdione, another major active ingredient of the essential oil [5]. Subsequently, curcumol became the key biological compound in Rhizoma Curcumae, and its bioactivity had recently attracted more attention. Curcumol, 5 – 7-membered guaianetype sesquiterpenes (Figure 1), was isolated from the rhizome of zedoary (Curcuma zedoaria Roscoe) firstly by Hiroshi in 1965 [6]. It has been reported to possess anti-tumor activity against a broad range of human cancer cells with low cytotoxicity. Curcumol can induce apoptosis and inhibit the proliferation of many kinds of tumor cells, such as gastric cancer, nasopharyngeal carcinoma, cervical cancer, and lung carcinoma cells [7 – 9]. But the significant inhibition of prolifer-
*Corresponding authors. Email: [email protected]; [email protected] q 2013 Taylor & Francis
54
P. Guo et al. derivative of curcumol, had been synthesized, and its structure was confirmed by single-crystal X-ray diffraction in our previous works [10]. The compound that had been isolated from the microbial oxidation products of curcumol [11] could be obtained easily through an improved chemical method. To study the structure –activity relationship, its analogs had been synthesized (Scheme 1). These derivatives of curcumol were assayed for their anti-tumor activity against gastric cancer cell lines SGC7901 and lung carcinoma cell line H460 (Table 1). The weak activity of compound 1 indicated that the special cyclic structure is important to combine with its biological target. By contrasting the activity of monoester and diester, the free 8-hydroxy is necessary to retain its anti-tumor activity. In the presence of electronwithdrawing phenyl group or amino group in the chain, many compounds showed outstanding efficiency to inhibit cell proliferation. The abnormal activity of compound 5 is possible due to its large stereospecific blockade. To improve the anti-tumor activity further, it is necessary to maintain the same cyclic structure and free 8-hydroxyl and meanwhile to import nitrogen atom into the structure (Figure 2).
H
O
OH
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Figure 1. The chemical structure of curcumol.
ation cannot be observed currently when the cells were dealed with curcumol under the concentration of 50 mg/ml. Obviously, it lacks enough efficiency to become a clinical drug. To improve its anti-tumor activity, a series of derivatives were synthesized starting from curcumol that was extracted from the volatile oil of Rhizoma Curcumae.
2.
Results and discussion
Rhizoma Curcumae oil (commercially available) was distilled in vacuum to remove volatile component with low boiling point. The residue was extracted repeatedly by petroleum ether. After removal of the extracted liquid, the viscous materials were crystallized in ethanol. Curcumol with 98% purity can be separated into 10–15% yield. In the molecular structure of curcumol, the number of H-bond donors and acceptors is little. The combination reaction between small molecular and corresponding biological target is not strong enough. It is unfavorable to exhibit the activity of curcumol. So, it is necessary to introduce more active groups such as hydroxyl and amino into the chemical structure of curcumol. Compound 1, a
3. Experimental 3.1 General experimental procedures Uncorrected melting points (m.p.) were determined using an X-4 (Gongyi, Henan, China) micro-melting point apparatus. 1H NMR and 13C NMR spectra were recorded OCOR
OH
H
1)m-CPBA O
Curcumol
OH
H
RCOCl O
2)NaOH 1
Scheme 1. Synthetic route of curcumol derivatives.
OH
or RCOOH
H O
OCOR
Journal of Asian Natural Products Research
solution to remove the residue m-CPBA. The organic phase was passed through anhydrous sodium sulfate to remove traces of water, and the solvent was evaporated under vacuum to obtain the mixture of epoxy derivatives. The mixture and solid NaOH were added into 20 ml ethanol, and the contents were refluxed for 2 h. To dump the solution into 100 ml water, product 1 was crystallized directly in 50% yield from this water. The synthetic route of product 2 was described in Ref. [10].
Table 1. The anti-tumor activity of curcumol derivatives.
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Compounds 1 2 3 4 5 6 7 8 Curcumol
SGC-7901 (mg/ml)
H460 (mg/ml)
175.3 168.7 . 200 . 200 34.7 37.7 24.0 120.8 72.6
196.4 173.6 . 200 . 200 47.8 40.5 17.1 150.3 78.5
3.3 General procedure for the preparation of diester derivatives
on a Bruker Avance-3600 (Bruker Technologies, Bremen, Germany) Spectrometer (600 MHz). Samples were dissolved in CDCl3, while tetramethylsilane (TMS) was used as an internal standard. Chemical shifts were recorded in (ppm) values relative to TMS, and J values were expressed in Hertz. MS were recorded using an Agilent-1100 LC – MS Spectrometer (Agilent Technologies, Waldbronn, Germany). The relative chemical reagents were purchased from Aladdin-reagent (Shanghai, China).
3.2
To a solution of 1 in dichloromethane, 2– 3 drops of triethylamine were added. The respective acid chloride dissolved in dichloromethane was added dropwise. The reaction mixture was stirred for 3 h with a magnetic stirrer, and the progress of the reaction was monitored by TLC. The products were purified by silica gel column chromatography using ethyl acetate and petroleum ether as the eluting solvent. The diester derivatives were obtained with 60 –80% yield.
Preparation of derivative 1
3.3.1
R1
14 2
15
10
1
9
O
3
Compound 3
A colorless oil, 1H NMR (CDCl3, 600 MHz) d: 0.82 (d, 3H, J ¼ 6.6 Hz, H-13), 0.85 (d, 3H, J ¼ 6.6 Hz, H-12), 0.93 (d, 3H, J ¼ 6.6 Hz, H-15), 1.13–1.25 (m, 1H, H6a), 1.41–1.46 (m, 1H, H-11), 1.50–1.55 (m, 1H, H-2a), 1.54–1.58 (m, 1H, H-3a), 1.74–1.77 (m, 1H, H-7), 1.76–1.80 (m, 1H, H-4), 1.79–1.84 (m, 1H, H-3b), 1.90–1.94
Curcumol (2.36 g) dissolved in 30 ml dichloromethane reacted with m-CPBA (3-chloroperbenzoic acid, 2.58 g) at room temperature. The progress of the reaction was monitored by thin-layer chromatography (TLC). After completion of the reaction, the reaction liquid was washed by sodium sulfite and sodium hydroxide
H
8
45 6
55
7
R2 13 11
Compound 1: R1=R2=OH; 2: R1=I, R2=OH; 3: R1=R2=CH3COO4: R1=R2=4-methyl-benzoyloxy; 5: R1=4-nitro-benzoyloxy, R2=OH 6: R1=4-chloro-benzoyloxy, R2=OH; 7: R1=Boc-Glycine, R2=OH 8: R1=Boc-Phenylalanine, R2=OH
Figure 2. Structures of the tested curcumol derivatives.
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(m, 1H, H-2b), 1.98 (s, 3H, ZCOCH3), 2.00 (s, 3H, ZCOCH3), 2.02–2.06 (m, 1H, H-1), 2.09 (m, 1H, H-6b), 4.41 (d, 1H, J ¼ 13.2 Hz, H-14), 4.47 (d, 1H, J ¼ 13.2 Hz, H-14), 5.81 (s, 1H, H-9); ESI-MS: m/z 359.1 [M þ Na]þ (100%). 13 C NMR (CDCl3, 150.9 MHz) d: 11.5 (C-15), 20.8 (ZOCOCH3), 21.1 (C-12), 22.0 (ZOCOCH3), 22.4 (C-13), 27.4 (C-2), 30.3 (C-11), 31.0 (C-3), 34.9 (C-6), 40.1 (C-4), 49.5 (C-1), 57.3 (C-7), 65.6 (C-14), 88.7 (C-5), 105.2 (C-8), 125.9 (C-9), 136.1 (C-10), 168.3 (ZOCO), 170.6 (ZOCO). 3.3.2
Compound 4
diimide was added. After stirring the mixture for 30 min, an equimolecular quantity of compound 1 was added. The products were separated from the mixture of reactions by silica gel column chromatography with 40– 60% yield. In the HMBC spectra of these compounds, significant correlations from proton H-14 to the involved carbonyl carbon could be observed. Obvious downfield shift of H-14 indicated that C-14 was linked with the ester group. These findings confirmed that esterification occurred only at the site of 14-OH. 3.4.1
1
A colorless oil, H NMR (CDCl3 , 600 MHz) d: 0.95 (d, 3H, J ¼ 6.6 Hz, H13), 1.02 (d, 3H, J ¼ 9.0 Hz, H-12), 1.03 (d, 3H, J ¼ 6.6 Hz, H-15), 1.24 –1.30 (m, 1H, H-6a), 1.59– 1.64 (m, 1H, H-11), 1.64– 1.69 (m, 1H, H-2a), 1.67 –1.72 (m, 1H, H3a), 1.80 – 1.84 (m, 1H, H-7), 1.92 – 1.97 (m, 1H, H-4), 1.90– 1.94 (m, 1H, H-3b), 1.88 –1.92 (m, 1H, H-2b), 2.10– 2.15 (m, 1H, H-1), 2.23– 2.28 (m, 1H, H-6b), 2.40 (s, 3H, ZPhZCH 3), 2.42 (s, 3H, ZPhZCH3), 4.80 (d, 1H, J ¼ 13.2 Hz, H14), 4.81 (d, 1H, J ¼ 13.2 Hz, H-14), 6.08 (s, 1H, H-9), 7.22 –7.27 (m, 4H, PhZH), 7.93 –8.01 (m, 4H, PhZH); ESI-MS: m/z 511.2 [M þ Na]þ (100%). 13 C NMR (CDCl3, 150.9 MHz) d: 11.8 (C-15), 21.8 (ZPhZCH3), 21.8 (ZPhZCH3), 21.9 (C-12), 22.7 (C-13), 27.9 (C-2), 30.6 (C-11), 31.2 (C-3), 35.2 (C-6), 40.4 (C-4), 50.0 (C-1), 58.4 (C-7), 66.4 (C-14), 89.2 (C-5), 105.7 (C-8), 126.5 (Ph-10 ), 126.8 (Ph-100 ), 129.3 (Ph-30 ,50 ), 129.6 (Ph-300 ,500 ), 130.1 (Ph-20 ,60 ), 130.4 (Ph-200 ,600 ), 136.6 (C-9), 143.8 (Ph-40 ), 143.9 (Ph-400 ), 144.7 (C-10), 164.1 (ZOCO), 166.5 (ZOCO). 3.4 General procedure for the preparation of monoester derivatives When the acid was dissolved in dichloromethane, excess of 1,3-dicyclohexylcarbo-
Compound 5
A yellow solid, m.p. 113–1168C; 1H NMR (CDCl3, 600 MHz) d: 0.85 (d, 3H, J ¼ 6.6 Hz, H-13), 0.96 (d, 3H, J ¼ 9.0 Hz, H-15), 0.98 (d, 3H, J ¼ 6.6 Hz, H-12), 1.22– 1.25 (m, 1H, H-6a), 1.44–1.46 (m, 1H, H11), 1.56–1.57 (m, 1H, H-2a), 1.59–1.60 (m, 1H, H-3a), 1.67–1.69 (m, 1H, H-7), 1.82–1.83 (m, 1H, H-4), 1.87–1.89 (m, 1H, H-3b), 1.89–1.91 (m, 1H, H-2b), 2.02–2.18 (m, 1H, H-1), 2.21 (dd, 1H, J ¼ 13.2 Hz, 10.8 Hz, H-6b), 4.75 (d, 1H, J ¼ 13.2 Hz, H14), 4.82 (d, 1H, J ¼ 13.2 Hz, H-14), 5.93 (s, 1H, H-9), 8.17 (d, 2H, J ¼ 9.0 Hz, PhZH), 8.26 (d, 2H, J ¼ 9.0 Hz, PhZH). 13 C NMR (CDCl3, 150.9 MHz) d: 11.7 (C-15), 21.8 (C-12), 22.6 (C-13), 27.6 (C-2), 30.6 (C-11), 31.2 (C-3), 36.4 (C-6), 40.2 (C4), 50.0 (C-1), 59.4 (C-7), 67.0 (C-14), 87.3 (C-5), 103.3 (C-8), 123.8 (Ph-30 ,50 ), 128.0 (C-9), 130.9 (Ph-20 ,60 ), 135.5 (Ph-10 ), 138.1 (C-10), 150.6 (Ph-40 ), 164.3 (CvO); ESIMS: m/z 402.3 [M þ H]þ (100%). 3.4.2
Compound 6
A white solid, m.p. 105 –1068C, 1H NMR (CDCl3, 600 MHz) d: 0.87 (d, 3H, J ¼ 6.6 Hz, H-13), 0.98 (d, 3H, J ¼ 6.6 Hz, H-12), 1.01 (d, 3H, J ¼ 6.6 Hz, H-15), 1.24 (m, 1H, H-6a), 1.48 – 1.50 (m, 1H, H11), 1.55– 1.59 (m, 1H, H-2a), 1.59– 1.64 (m, 1H, H-3a), 1.65 –1.68 (m, 1H, H-7), 1.82 –1.84 (m, 1H, H-4), 1.84 – 1.89 (m,
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Journal of Asian Natural Products Research 1H, H-2b), 1.90– 1.92 (m, 1H, H-3b), 2.03 –2.05 (m, 1H, H-1), 2.19 –2.23 (m, 1H, H-6b), 4.71 (d, 1H, J ¼ 13.2 Hz, H14), 4.78 (d, 1H, J ¼ 13.2 Hz, H-14), 5.93 (s, 1H, H-9), 7.40 (d, 2H, J ¼ 8.4 Hz, Ph-H), 7.95 (d, 2H, J ¼ 8.4 Hz, PhZH). 13 C NMR (CDCl3, 150.9 MHz) d: 11.8 (C-15), 21.5 (C-12), 22.7 (C-13), 27.7 (C2), 30.9 (C-11), 31.3 (C-3), 36.5 (C-6), 40.3 (C-4), 50.1 (C-1), 59.6 (C-7), 66.4 (C-14), 87.4 (C-5), 103.4 (C-8), 127.3 (C-9), 128.0 (Ph-10 ), 128.9 (Ph-30 ,50 ), 131.1 (Ph-20 ,60 ), 138.8 (C-10), 165.4 (CvO), 139.7 (Ph-40 ); ESI-MS: m/z 391.4 [M þ H]þ (100%). 3.4.3 Compound 7 A white solid, m.p. 88 –908C, 1H NMR (CDCl 3, 600 MHz) d: 0.84 (d, 3H, J ¼ 6.6 Hz, H-13), 0.95 (d, 3H, J ¼ 6.6 Hz, H-12), 0.98 (d, 3H, J ¼ 6.6 Hz, H-15), 1.18 –1.22 (m, 1H, H-6a), 139– 1.42 (m, 1H, H-11), 1.42 (d, 9H, J ¼ 1.2 Hz, ZC(CH3)3), 1.49– 1.51 (m, 1H, H-2a), 1.51– 1.55 (m, 1H, H-3a), 1.61 –1.66 (m, 1H, H-7), 1.78 –1.82 (m, 1H, H-4), 1.82– 1.86 (m, 1H, H-2b), 1.85– 1.89 (m, 1H, H-3b), 1.94 –1.98 (m, 1H, H1), 2.15 – 2.19 (m, 1H, H-6b), 3.90 (d, 2H, J ¼ 4.8 Hz, ZCH2NH), 4.51 (d, 1H, J ¼ 13.2 Hz, H-14), 4.58 (d, 1H, J ¼ 13.2 Hz, H-14), 5.82 (s, 1H, H-9), 3.60 (br, 1H, ZOH), 5.12 (br, 1H, ZNH). 13 C NMR (CDCl3, 150.9 MHz) d: 11.7 (C-15), 21.5 (C-12), 22.7 (C-13), 27.5 (C-2), 29.8 (Boc-Gly), 30.8 (C-11), 31.2 (C-3), 36.4 (C-6), 40.3 (C-4), 42.5 (Boc-Gly), 49.7 (C-1), 59.4 (C-7), 66.4 (C-14), 80.1 (Boc-Gly), 87.2 (C-5), 103.3 (C-8), 127.8 (C-9), 138.3 (C-10), 155.8 (Boc-Gly), 170.3 (Boc-Gly); ESI-MS: m/z 410.2 [M þ H]þ (100%). 3.4.4
Compound 8
A white solid, m.p. 74 –778C, 1H NMR (CDCl 3, 600 MHz) d: 0.86 (d, 3H, J ¼ 6.6 Hz, H-13), 0.98 (d, 3H, J ¼ 6.6 Hz, H-12), 1.00 (d, 3H, J ¼ 6.6 Hz, H-15), 1.16 – 1.21 (m, 1H, H-6b), 1.38 –1.41 (m, 1H, H-11), 1.41 (d, 9H,
57
J ¼ 1.2 Hz, ZC(CH 3) 3), 1.47 – 1.51 (m, 1H, H-2a), 1.53 – 1.58 (m, 1H, H3a), 1.62– 1.67 (m, 1H, H-7), 1.78– 1.82 (m, 1H, H-4), 1.77 –1.81 (m, 1H, H-2b), 1.84 –1.88 (m, 1H, H-3b), 1.88 –1.93 (m, 1H, H-1), 2.15 –2.20 (m, 1H, H-6a), 4.43 (d, 1H, J ¼ 13.2 Hz, H-14), 4.57 (d, 1H, J ¼ 13.2 Hz, H-14), 5.79 (s, 1H, H-9), 3.00 –3.12 (m, 2H, ZCH2Ph), 4.52– 4.63 (m, 1H, ZCHNH), 7.12 – 7.28 (m, 5H, PhZH), 3.30 (br, 1H, ZOH), 5.01 (br, 1H, ZNH). 13 C NMR (CDCl3, 150.9 MHz) d: 11.6 (C-15), 21.2 (C-12), 22.1 (C-13), 27.5 (C-2), 28.4 (Boc-Phe), 30.8 (C-11), 31.2 (C-3), 36.4 (C-6), 38.5 (Boc-Phe), 40.3 (C-4), 49.6 (C1), 54.6 (Boc-Phe), 59.5 (C-7), 66.5 (C-14), 80.0 (Boc-Phe), 87.1 (C-5), 103.3 (C-8), 125.5 (Boc-Phe), 127.1 (Boc-Phe), 127.8 (C9), 128.5 (Boc-Phe), 128.6 (Boc-Phe), 129.3 (Boc-Phe), 136.1 (Boc-Phe), 138.3 (C-10), 155.2 (Boc-Phe), 171.8 (Boc-Phe); ESI-MS: m/z 499.9 [M þ H]þ (100%).
3.5
Bioassay
To conduct the methyl thiazolyl tetrazolium (MTT) assay, the cells (3000 per well) were seeded in wells of a 96-well plate in 100 ml of complete media, and incubated for 3 days at 378C. Then 20 ml of MTT dissolved at 5 mg/ml in sterile phosphatebuffered saline was added to the cells. Four hours later, the media was removed, the metabolized MTT was suspended in dimethyl sulfoxide, and the absorbance was measured at 490 nm. The experiment was conducted in triplicates, and the statistical analysis (simple linear regression analysis initially and then unpaired Student’s test that revealed significant differences between two sample means) was carried out to obtain the final values.
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