Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
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Synthesis and biological evaluation of a novel class of coumarin derivatives Hong Li a, Xiaomin Wang b, Guichao Xu a, Li Zeng a, Kai Cheng a, Pengchao Gao c, Qing Sun a, Wei Liao a, Jianwei Zhang a,⇑ a b c
College of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR China Department of Physiology, Key Laboratory for Neurodegenerative Disorders of the Ministry of Education, Capital Medical University, Beijing 100069, PR China School of Pharmaceutical Sciences, Peking University, Beijing 100191, PR China
a r t i c l e
i n f o
Article history: Received 8 June 2014 Revised 11 September 2014 Accepted 18 September 2014 Available online xxxx Keywords: Amino acid Coumarin Peptide Anti-tumor Cytotoxicity
a b s t r a c t In this study, several novel coumarin derivatives, 7-hydroxy-2-oxo-2H-chromene-3-carboxyl-TrpTrp-AA-OBzl compounds, were designed and synthesized as potential anticancer agents. Their in vitro cytotoxic activities were evaluated using methylthiazoltetrazolium (MTT) assay. The anti-tumor activity of the newly coumarin derivatives was determined in a S180 bearing mouse model and some of the compounds demonstrated tumor growth inhibition similar to the positive control, doxorubicin. Compared to doxorubicin, most of the compounds exhibited enhanced immunologic function suggesting a relatively minor toxic effect. The intercalation of the coumarin derivatives synthesized with calf thymus (CT) DNA was also studied. Ó 2014 Elsevier Ltd. All rights reserved.
Cancer has long been one of the serious diseases threatening human health and continues to be a major health problem worldwide. Therefore, discovering new compounds with potent anticancer activity is of utmost importance. Among current anticancer chemotherapeutic agents DNA-recognizing molecules, including intercalating agents, alkylating compounds and groove binders, are especially interesting. DNA intercalating agents are important in clinical oncology, and several representative compounds (anthracyclines, anthraquinones and acridines) are conventionally used.1 Intercalation results in conformational alters of the double helix, and then changes the processes of DNA replication, transcription and repairing. Thus the discovery of novel DNA intercalating agents is considered as a promising approach toward anticancer drugs. Coumarins are the most important classes of natural products with a variety of pharmacological activity. Coumarin and Coumarin-related compounds have proved for many years to have significant therapeutic potential and are a plentiful source of potential drugs candidate in relation to its safety and efficacy. The bioactivity of coumarin and more complex related derivatives appears to be based on the coumarin nucleus.1 Biological effects observed include anti-bacterial,2 anti-thrombotic and vasodilatory,3
⇑ Corresponding author. Tel.: +86 10 8391 1522; fax: +86 10 8391 1533.
anti-mutagenic,4 lipoxygenase and cyclooxygenase inhibition,5,6 scavenging of reactive oxygen species, and anti-tumour.7–13 Previous studies on a variety of synthetic coumarin derivatives have demonstrated the influence of the coumarin skeleton and substitutions at Positions 3 and 7 on antitumor activities.14,15 Some studies demonstrated that coumarin derivatives containing a substituted hydroxy group at the Position 7 possess antibiotic and antifungal activities. The in vitro effects of coumarins on the growth of renal cell carcinoma-derived cell lines showed that coumarin and 7-hydroxycoumarin were potent cytotoxic and cytostatic agents.16 The hydroxyl group on the coumarin ring is crucial to its anti-cancer activity. Some studies demonstrated that hydroxylated derivatives containing a substituted carboxylic group at Position 3 were capable of decreasing cancer cell viability and inhibiting DNA synthesis. It is well proved that Trp-Trp is biologically important either as a dipeptide or as a fragment of some peptides. Trp-Trp-OBz was used as a lead, and twenty tripeptide benzyl esters, Trp-Trp-AAOBz, were synthesized as DNA intercalators.17 Coumarin-3-carboxamides were reported to exhibit selective cytotoxicity against mammalian cancer cell lines and as inhibitors of serine proteases, a-chymotrypsin (CT) and human leukocyte elastase (HLE).18 In view of the importance associated with the above cited moieties in their potent anti-tumor activity, it was thought of considerable interest to synthesize derivatives of Trp-Trp-AA-OBz for the generation of new 7-hydroxy-2-oxo-2H-chromene-3-carboxyl
E-mail address: [email protected] (J. Zhang). http://dx.doi.org/10.1016/j.bmcl.2014.09.051 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
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coumarins which are represented in the Table 1. In addition, the use of tripeptide in coumarin derivatives designed was based upon the concept that many amino acids with functional side chains are capable of making base-specific contacts with more than one type of DNA bases.19 Moreover, amino acid conjugates might target the gastrointestinal transporters involved in the absorption of amino acids and small peptides resulting in improved oral bioavailability.20,21 The various side chains of different amino acids allow the addition of amino acids to coumarin to manipulate the pharmacokinetics profiles of the compounds. In addition, various amino acids can be introduced to enhance solubility. Therefore, in order to search for better anti-tumor agents, coumarin was used as the basic molecule and a series of novel 7-hydroxycoumarin derivatives bearing Trp-Trp-AA-OBzl at Position 3 were designed and synthesized and evaluated for their anti-tumor activity in the present study. As shown in Scheme 2, the syntheses of coumarin derivatives were carried out using a multi-step synthetic route. Firstly, ethyl 7-hydroxy-2-oxo-2H-chromene-3-carboxylate (3) was synthesized via the Knoevenagel condensation of 2,4-dihydroxybenzaldehyde (1) with diethyl malonate (2) in piperidine in an almost 90% yield. Subsequently, removal of the ethyl group with refluxing 4M hydrochloric acid resulted in 7-hydroxy-2-oxo-2H-chromene-3-carboxylic acid (4).22 Finally, tripeptide benzyl esters, NH2-Trp-Trp-AAOBzl, from Scheme 1 were introduced into 4 by the DCC/HOBt/Nmethylmorpholine (NMM) procedure to provide the respective coumarin derivatives, 7-hydroxy-2-oxo-2H-chromene-3-carboxyl-Trp-Trp-AA-OBzl (5–17), as shown in Scheme 2 (yields, 59–84%). The chemical structures of all coumarin derivatives (3– 17) are provided in Table 1 and were confirmed by 1H NMR, 13C NMR, IR, and HR-ESIMS. The spectroscopic data are given in the ‘Supporting information’ Section. The 1H NMR spectra of coumarin derivatives showed signals at d 8.67–8.78 for one proton corresponding to H-4 of the coumarin skeleton. The two pairs of 1H double doublet at d 4.72–4.87, 4.62–4.77 are in agreement with amethine proton of tryptophan. Signals at 4.72–4.87 and 4.62– 4.77 in the COSY spectrum indicated methylenes adjacent to
Boc-Trp-OH+ NH2-Trp-OMe iii
Boc-Trp-Trp-AA-OBzl
i
ii
Boc-Trp-Trp-OMe
iv
Boc-Trp-Trp-OH
NH2 -Trp-Trp-AA-OBzl
Scheme 1. Preparation of NH2-Trp-Trp-AA-OBzl. Reagents and conditions: (i) NMM, DCC, HOBt; (ii) 2 M NaOH; (iii) NH2-AA-OBzl, NMM, DCC, HOBt; (iv) hydrogen chloride in ethyl acetate (4 mol/L).
HO
OH C
+
H
O O
O 1
O
O
O
O
O
O CO 2Et
3
2
HO
ii
HO
i
HO
O
O
iii
COTrp-Trp-AA-OBzl
CO2 H 5-17
4
Scheme 2. Synthesis of 7-hydroxy-2-oxo-2H-chromene-3-carboxylic acid-Trp-TrpAA-OBzl compounds. Reagents and conditions: (i) diethyl malonate and piperidine; (ii) HCl; (iii) NH2-Trp-Trp-AA-OBzl, NMM, DCC, HOBt.
methines. 1H NMR chemical shifts values at d 3.22–2.87 suggested the presence of methylene protons of tryptophan. 1H NMR spectroscopic signals at d 4.68–3.94 are in agreement with a-methine proton of the AA in NH2-Trp-Trp-AA-OBzl. The in vitro cytotoxicity of the coumarin derivatives synthesized above was evaluated in human lung adenocarcinoma cells (A549), chronic myeloid leukemia cells (K562), human liver carcinoma cells (HepG2), Human Glioblastoma cells (A172), and human hepatocellular carcinoma cells (Bel-7402) using MTT assay.23 Doxorubicin (adriamycin, ADM) was used as positive control. In brief, cells were exposed to 4–17 of concentrations ranging from
Table 1 Structures of coumarin derivatives synthesized Compound No.
Chemical structure HO
O
Compound No.
O
Chemical structure
HO
O
HO
O
O
4
3 CO2Et
HO
O
CO2H
O
5
O
6
COTrp-Trp-Asp(OBzl)-OBzl
COTrp-Trp-OBzl
HO
O
O
7
HO
O
HO
O
O
8
COTrp-Trp-Gly-OBzl
HO
COTrp-Trp-Phe-OBzl
O
O
9
O
10
COTrp-Trp-Leu-OBzl HO
O
COTrp-Trp-Ile-OBzl
O
11
HO
O
HO
O
HO
O
O
12
COTrp-Trp-Val-OBzl HO
O
COTrp-Trp-Ser-OBzl
O
13
O
14
COTrp-Trp-Tyr-OBzl
COTrp-Trp-Thr-OBzl
HO
O
O
15
O
16
COTrp-Trp-Pro-OBzl HO
O
COTrp-Trp-Trp-OBzl
O
17 COTrp-Trp-Glu(OBzl)-OBzl
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1 lM to 200 lM for 48 h and cell survival was then determined. As shown in Table 2, the IC50 values of ADM in HepG2, K562, A172, Bel-7402 and A549 cells were found to be 10.1, 8.9, 18.5, 11.7 and 4.3 lM, respectively. 4–17 exhibited varied activities in the five cell lines and they were found most effective in K562 cells and least effective in Bel-7402 cells. In K562 cells, 5, 7, 9, 14, 15 and 16 were found to be most effective with IC50 values of lower than 30 lM. The IC50 of 9 (IC50, 14.7 lM) is lower than that of 10 (IC50, >200.0 lM) suggesting that the methyl group at the b or c position of an amino acid residue cause changes in the cytotoxicity. Compared to 8 (IC50, 70.3 lM), 14 (IC50, 19.6 lM) exhibited a higher cytotoxicity suggesting that the introduction of the additional hydroxyl group in the phenylalanine increased the cytotoxicity. In A172 cells, 7, 9 and 10 showed potent cytotoxic activity with IC50 values of lower than 27.3 lM and Compound 7 demonstrated the significant cytotoxic activity with an IC50 value of 10.1 lM and was better than the positive control doxorubicin ADM (IC50, 18.5 lM). In HepG2 cells, 7 was found to be most effective with an IC50 value of 16.4 lM. In A549 cells, 8 and 9 were found to be most effective with IC50 values of lower than 29.2 lM. The coumarin derivatives were found to be generally less potent in Bel-7402 cells. Among all the compounds 6 was found to be the most potent compounds of this series with an IC50 value of 45.5 lM, 5 and 9 displayed the prominent and broader spectrum of cytotoxic activities against all the five human tumor cell lines tested with four IC50 values lower than 49.2 lM and 22.9 lM, respectively. The results seem to suggest that the amino acid in the Trp-Trp-AA-OBzl at position 3 of coumarin ring plays an important role in the cytotoxicity observed. The anti-tumor activity of coumarin derivatives synthesized (4– 17) was evaluated in mice bearing S180.24 The mice were given a daily ip injection of 1 lmol/kg of 4–17 in 0.2 mL of normal saline (NS) for seven consecutive days with NS as the negative control. ADM at 1 lmol/kg (0.2 mL) dissolved in 0.9% saline was used as positive control. The tumor weights were found to be 1.05–0.56 g compared to 1.45 g in the negative control group. The tumor inhibition % in describing the anti-tumor effects was determined as:
Tumor Inhibition% ¼ ðC TÞ=C 100 T: average tumor weight of treated group; C: average tumor weight of negative control group The anti-tumor activity results of these compounds (4–17) are summarized in Table 3. The tumor inhibition % of the compounds tested were found to range from 27.3% (17) to 61.5% (14). Comparison of inhibition ratio values of Compound 4 (34.2%) and Compound 5 (48.0%) showed that the presence of Trp-Trp dipeptide
Table 2 IC50 values of 4–17 in K562, A172, BL-7402, A549 and HepG2 cell lines
a
Compoundsa
HepG2
K562
A172
Bel-7402
A549
4 5 6 7 8 9 10 11 12 13 14 15 16 17 ADM
>200.0 33.1 ± 0.9 72.4 ± 1.2 16.4 ± 0.5 >200.0 22.9 ± 0.7 104.5 ± 1.7 >200.0 76.7 ± 1.4 33.5 ± 0.6 59.4 ± 0.9 >200.0 >200.0 >200.0 10.1 ± 1.2
>200.0 29.1 ± 1.3 >200.0 23.9 ± 0.9 70.3 ± 1.4 14.7 ± 1.3 >200.0 >200.0 >200.0 32.9 ± 1.0 19.6 ± 1.1 27.6 ± 1.7 28.0 ± 0.9 >200.0 8.9 ± 0.3
>200.0 99.5 ± 1.0 67.8 ± 1.3 10.1 ± 0.6 147.5 ± 2.9 21.9 ± 0.6 26.0 ± 1.1 58.1 ± 0.9 165.7 ± 1.6 63.4 ± 0.9 35.4 ± 0.4 90.7 ± 0.9 >200.0 >200.0 18.5 ± 0.7
>200.0 49.2 ± 1.4 45.5 ± 0.9 100.0 ± 1.4 >200.0 94.9 ± 1.0 95.5 ± 1.6 49.1 ± 0.5 >200.0 98.1 ± 1.3 >200.0 >200.0 >200.0 >200.0 11.7 ± 0.7
>200.0 45.9 ± 0.6 >200.0 >200.0 29.2 ± 1.2 22.0 ± 0.9 49.1 ± 2.1 194.2 ± 0.9 >200.0 >200.0 71.0 ± 0.9 >200.0 >200.0 >200.0 4.3 ± 0.4
IC50 value is expressed by X SD lM and n = 6.
Table 3 Tumor inhibition % by 4–17 in S180 bearing mice Compounda
Dose (lmol/kg)
Tumor weight (g)
Tumor inhibition %
NS ADM 4 5 6 7 8 9 10 11 12 13 14 15 16 17
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1.45 ± 0.29 0.41 ± 0.08 0.95 ± 0.10b 0.75 ± 0.07b 0.74 ± 0.14b 0.59 ± 0.12b 0.73 ± 0.13b 0.65 ± 0.09b 0.77 ± 0.10b 0.75 ± 0.13b 0.84 ± 0.13b 0.93 ± 0.15b 0.56 ± 0.11b 0.77 ± 0.14b 0.72 ± 0.11b 1.05 ± 0.19c
71.7 ± 5.6 34.2 ± 7.1 48.0 ± 7.7 49.0 ± 9.6 59.5 ± 8.8 50.0 ± 9.0 55.1 ± 7.4 46.8 ± 6.9 48.4 ± 9.5 42.2 ± 8.2 35.9 ± 5.0 61.5 ± 8.3 47.2 ± 9.7 50.4 ± 0.9 27.3 ± 4.2
a ADM = Positive control, NS = Vehicle, n = 12, tumor weight is expressed by X SD g. b Compared to NS p < 0.01. c Compared to NS p < 0.05.
can increased the activity, suggesting that the Trp-Trp dipeptide structure had some contribution to the activity. Compounds 7 and 14 showed the highest tumor inhibition (59.5% and 61.5%, respectively) in mice bearing S180. Compared to 8 (50.0%), 14 had prominent anti-tumor activities (61.5%). It implied that the free hydroxyl group of tyrosine might play a vital role in the anti-tumor activity. Compared to 9 (55.1%), 10 and 11 exhibited a lower activity (46.8% and 48.4%, respectively) suggesting that the methyl group at the b position and the isopropyl group at the a position of an amino acid residue is likely responsible for the reduced activity observed. Compound 7 without substituent at position-a exhibited potent anti-tumor activity (59.5%). Since the coumarin derivatives showed higher degree of tumor inhibition, the preliminary toxicity of 4–17 were evaluated during the administration. All the tested compounds caused neither obvious neurotoxic reaction including tremor, twitch, jumping, tetanus and supination nor death at tested dosage. Twenty-four hours after the last administration the mice were decapitated. Necropsy of the dead mice was carried out. The necropsy revealed no apparent changes in any organs. Brain, heart, liver, spleen and kidney were weighed. The spleen indexes were measured. Most of the tested compounds exhibited a relatively minor toxic effect or better than the positive control, ADM. The data are listed in Table 4. Besides, 7 was examined for the LD50. The results indicate that even the dose of 7 was up to 500 mg/kg the mice occurred no death. These suggest that 7 is comparatively non-toxic and its LD50 values should be more than 500 mg/kg. Since two structural elements coumarin and Trp-Trp of the coumarin derivatives synthesized, each has a planar polycyclic aromatic pharmacophore capable of stacking between DNA pairs at the intercalation sites,25 the intercalation of the coumarin derivatives with calf thymus (CT) DNA was investigated by UV absorption, fluorescence and circular dichroism (CD) spectroscopy using 7. The UV spectrum of CT DNA in PBS (pH 7.4, 160 lM) was recorded on a Shimadzu 2550 spectrophotometer from 200 to 350 nm. 2 mL of CT DNA in PBS was then titrated with 20 lL of Compound 7 in PBS (pH 7.4) solutions of 0, 2.0, 4.0, 6.0, 8.0 and 10.0 lM, respectively. As shown in Figure 1, the UV absorption of CT DNA gradually decreased from 1.052 to 0.974 with the increase of 7 concentration and a hypochromic effect (7.4%) was induced.26 The fluorescence spectrum of 7 in PBS (pH 7.4, 14 lM) was obtained and compared with that in the presence of CT DNA in PBS (final concentrations: 0, 1.5, 3.0, 4.5, 6.0 and 7.5 lM, respectively,
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Table 4 Effect of 4–17 on the organs weight and spleen index of S180 mice
a b c
Compounda
Brain weight (g)
Heart weight (g)
Liver weight (g)
Kidney weight (g)
Spleen index
NS ADM 4 5 6 7 8 9 10 11 12 13 14 15 16 17
0.30 ± 0.02 0.29 ± 0.02 0.29 ± 0.04 0.28 ± 0.03 0.31 ± 0.04 0.29 ± 0.03 0.27 ± 0.05 0.28 ± 0.05 0.24 ± 0.07b 0.26 ± 0.06 0.26 ± 0.05 0.29 ± 0.04 0.28 ± 0.05 0.24 ± 0.06b 0.25 ± 0.05b 0.26 ± 0.05c
0.12 ± 0.02 0.11 ± 0.01 0.12 ± 0.01 0.10 ± 0.01 0.13 ± 0.02 0.11 ± 0.02 0.11 ± 0.02 0.11 ± 0.01 0.10 ± 0.01 0.09 ± 0.01b 0.12 ± 0.02 0.12 ± 0.02 0.10 ± 0.02 0.09 ± 0.02b 0.12 ± 0.03 0.11 ± 0.01
1.84 ± 0.32 1.60 ± 0.31c 1.81 ± 0.40 1.73 ± 0.33 1.61 ± 0.26c 1.86 ± 0.44 1.78 ± 0.44 1.60 ± 0.39c 1.78 ± 0.34 1.78 ± 0.31 1.90 ± 0.36 1.82 ± 0.41 1.76 ± 0.26 1.87 ± 0.32 1.80 ± 0.35 1.85 ± 0.32
0.16 ± 0.04 0.16 ± 0.04 0.20 ± 0.02 0.14 ± 0.04 0.16 ± 0.04 0.20 ± 0.04 0.17 ± 0.04 0.18 ± 0.04 0.17 ± 0.04 0.16 ± 0.03 0.18 ± 0.04 0.17 ± 0.03 0.15 ± 0.05 0.14 ± 0.03 0.17 ± 0.04 0.18 ± 0.03
7.25 ± 1.60 5.76 ± 0.99b 8.44 ± 1.93 7.72 ± 1.72 5.92 ± 1.52b 7.92 ± 1.96 8.69 ± 2.12 5.41 ± 1.32b 7.94 ± 1.58 6.97 ± 1.44 8.15 ± 1.97 7.43 ± 1.57 7.12 ± 1.55 4.67 ± 1.04b 6.93 ± 1.36 7.34 ± 1.40
Dose of ADM and 4–17: 1 lmol/kg, ADM = Positive control, NS = Vehicle, n = 12, spleen index is expressed by X SD mg/kg body weight. Compared to NS p < 0.01. Compared to NS p < 0.05.
Figure 1. UV spectra of CT DNA (pH = 7.4, concentration 160 lM) in the absence and presence of 7 of 0, 2.0, 4.0, 6.0, 8.0 and 10.0 lM, respectively.
Figure 3. CD spectra of CT DNA (pH = 7.4, concentration 160 lM) in the absence and presence of 7 (concentration 100 lM).
Figure 2. Fluorescence spectra of 7 (concentration 14 lM) in the absence and presence of CT DNA (pH = 7.4) of 0, 1.5, 3.0, 4.5, 6.0 and 7.5 lM, respectively.
pH 7.4) on a Shimadzu RF-5310PC spectrofluorometer (excitation wavelength 401 nm and emission wave-length 447 nm).27 When the concentration of CT DNA was increased to 7.5 lM the fluorescence intensity of Compound 7 was decreased by 15.9% from 536.7 to 451.2 as shown in Figure 2. In our CD experiments a solution of CT DNA alone in PBS buffer and a solution of CT DNA plus the representative 7 in PBS buffer were incubated at 37 °C for 24 h, and then their CD spectra were obtained in Figure 3.28 The results revealed the effect of intercalation of 7 into base stacking. This study provided some useful information to understand the interaction mechanism at the molecular level. In summary, 7-hydroxycoumarin derivatives bearing Trp-TrpAA-OBzl at Position 3 were designed, synthesized and evaluated. In vitro cytotoxicity assays against five human carcinoma cell lines (K562, A172, BL-7402, A549 and HepG2) explored the cell selective anti-proliferation for individual compounds. The in vivo anti-tumor activity assays with S180 mice revealed 7 and 14 having the highest efficacy. The tumor inhibition % of 7 and 14 is similar to the positive control doxorubicin ADM. The UV and fluorescence spectra, as well as and circular dichroism spectroscopy of 7 with or without CT DNA suggest that the binding of the coumarin derivatives synthesized to
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DNA was intercalative. These compounds may be considered as a structural clue for the further studies. Acknowledgments This work was supported by the Academic Human Resources Development in Institutions of Higher Learning Under the Jurisdiction of Beijing Municipality (PHR201007114), the National Basic Research Program of China (2011CB504100), the National Natural Scientific Foundation of China (81030062) and Key National Science & Technology Specific Projects (2011ZX09102-003-01), Beijing Pharmacological Society (2013BI-01). Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.09. 051. References and notes 1. Jimenez-Orozco, F. A.; Molina-Guarneros, J. A.; Mendoza-Patino, N.; LeonCedeno, F.; Flores-Perez, B.; Santos-Santos, E.; Mandoki, J. J. Melanoma Res. 1999, 9, 243. 2. Laurin, P.; Ferroud, D.; Klich, M.; Dupis-Hamelin, C.; Mauvais, P.; Lassaigne, P.; Bonnefoy, A.; Musicki, B. Bioorg. Med. Chem. Lett. 1999, 9, 2079. 3. Hoult, J. R. S.; Paya, M. Gen. Pharmacol. 1996, 27, 713. 4. Pillai, S. P.; Menon, S. R.; Mitscher, L. A.; Pillai, C. A.; Shankel, D. M. J. Nat. Prod. 1999, 62, 1358. 5. Kimura, Y.; Okuda, H.; Arichi, S.; Baba, K.; Kozawa, M. Biochim. Biophys. Acta 1985, 834, 224.
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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of a novel class of coumarin derivatives Hong Li a, Xiaomin Wang b, Guichao Xu a, Li Zeng a, Kai Cheng a, Pengchao Gao c, Qing Sun a, Wei Liao a, Jianwei Zhang a,⇑ a b c
College of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR China Department of Physiology, Key Laboratory for Neurodegenerative Disorders of the Ministry of Education, Capital Medical University, Beijing 100069, PR China School of Pharmaceutical Sciences, Peking University, Beijing 100191, PR China
a r t i c l e
i n f o
Article history: Received 8 June 2014 Revised 11 September 2014 Accepted 18 September 2014 Available online xxxx Keywords: Amino acid Coumarin Peptide Anti-tumor Cytotoxicity
a b s t r a c t In this study, several novel coumarin derivatives, 7-hydroxy-2-oxo-2H-chromene-3-carboxyl-TrpTrp-AA-OBzl compounds, were designed and synthesized as potential anticancer agents. Their in vitro cytotoxic activities were evaluated using methylthiazoltetrazolium (MTT) assay. The anti-tumor activity of the newly coumarin derivatives was determined in a S180 bearing mouse model and some of the compounds demonstrated tumor growth inhibition similar to the positive control, doxorubicin. Compared to doxorubicin, most of the compounds exhibited enhanced immunologic function suggesting a relatively minor toxic effect. The intercalation of the coumarin derivatives synthesized with calf thymus (CT) DNA was also studied. Ó 2014 Elsevier Ltd. All rights reserved.
Cancer has long been one of the serious diseases threatening human health and continues to be a major health problem worldwide. Therefore, discovering new compounds with potent anticancer activity is of utmost importance. Among current anticancer chemotherapeutic agents DNA-recognizing molecules, including intercalating agents, alkylating compounds and groove binders, are especially interesting. DNA intercalating agents are important in clinical oncology, and several representative compounds (anthracyclines, anthraquinones and acridines) are conventionally used.1 Intercalation results in conformational alters of the double helix, and then changes the processes of DNA replication, transcription and repairing. Thus the discovery of novel DNA intercalating agents is considered as a promising approach toward anticancer drugs. Coumarins are the most important classes of natural products with a variety of pharmacological activity. Coumarin and Coumarin-related compounds have proved for many years to have significant therapeutic potential and are a plentiful source of potential drugs candidate in relation to its safety and efficacy. The bioactivity of coumarin and more complex related derivatives appears to be based on the coumarin nucleus.1 Biological effects observed include anti-bacterial,2 anti-thrombotic and vasodilatory,3
⇑ Corresponding author. Tel.: +86 10 8391 1522; fax: +86 10 8391 1533.
anti-mutagenic,4 lipoxygenase and cyclooxygenase inhibition,5,6 scavenging of reactive oxygen species, and anti-tumour.7–13 Previous studies on a variety of synthetic coumarin derivatives have demonstrated the influence of the coumarin skeleton and substitutions at Positions 3 and 7 on antitumor activities.14,15 Some studies demonstrated that coumarin derivatives containing a substituted hydroxy group at the Position 7 possess antibiotic and antifungal activities. The in vitro effects of coumarins on the growth of renal cell carcinoma-derived cell lines showed that coumarin and 7-hydroxycoumarin were potent cytotoxic and cytostatic agents.16 The hydroxyl group on the coumarin ring is crucial to its anti-cancer activity. Some studies demonstrated that hydroxylated derivatives containing a substituted carboxylic group at Position 3 were capable of decreasing cancer cell viability and inhibiting DNA synthesis. It is well proved that Trp-Trp is biologically important either as a dipeptide or as a fragment of some peptides. Trp-Trp-OBz was used as a lead, and twenty tripeptide benzyl esters, Trp-Trp-AAOBz, were synthesized as DNA intercalators.17 Coumarin-3-carboxamides were reported to exhibit selective cytotoxicity against mammalian cancer cell lines and as inhibitors of serine proteases, a-chymotrypsin (CT) and human leukocyte elastase (HLE).18 In view of the importance associated with the above cited moieties in their potent anti-tumor activity, it was thought of considerable interest to synthesize derivatives of Trp-Trp-AA-OBz for the generation of new 7-hydroxy-2-oxo-2H-chromene-3-carboxyl
E-mail address: [email protected] (J. Zhang). http://dx.doi.org/10.1016/j.bmcl.2014.09.051 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
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coumarins which are represented in the Table 1. In addition, the use of tripeptide in coumarin derivatives designed was based upon the concept that many amino acids with functional side chains are capable of making base-specific contacts with more than one type of DNA bases.19 Moreover, amino acid conjugates might target the gastrointestinal transporters involved in the absorption of amino acids and small peptides resulting in improved oral bioavailability.20,21 The various side chains of different amino acids allow the addition of amino acids to coumarin to manipulate the pharmacokinetics profiles of the compounds. In addition, various amino acids can be introduced to enhance solubility. Therefore, in order to search for better anti-tumor agents, coumarin was used as the basic molecule and a series of novel 7-hydroxycoumarin derivatives bearing Trp-Trp-AA-OBzl at Position 3 were designed and synthesized and evaluated for their anti-tumor activity in the present study. As shown in Scheme 2, the syntheses of coumarin derivatives were carried out using a multi-step synthetic route. Firstly, ethyl 7-hydroxy-2-oxo-2H-chromene-3-carboxylate (3) was synthesized via the Knoevenagel condensation of 2,4-dihydroxybenzaldehyde (1) with diethyl malonate (2) in piperidine in an almost 90% yield. Subsequently, removal of the ethyl group with refluxing 4M hydrochloric acid resulted in 7-hydroxy-2-oxo-2H-chromene-3-carboxylic acid (4).22 Finally, tripeptide benzyl esters, NH2-Trp-Trp-AAOBzl, from Scheme 1 were introduced into 4 by the DCC/HOBt/Nmethylmorpholine (NMM) procedure to provide the respective coumarin derivatives, 7-hydroxy-2-oxo-2H-chromene-3-carboxyl-Trp-Trp-AA-OBzl (5–17), as shown in Scheme 2 (yields, 59–84%). The chemical structures of all coumarin derivatives (3– 17) are provided in Table 1 and were confirmed by 1H NMR, 13C NMR, IR, and HR-ESIMS. The spectroscopic data are given in the ‘Supporting information’ Section. The 1H NMR spectra of coumarin derivatives showed signals at d 8.67–8.78 for one proton corresponding to H-4 of the coumarin skeleton. The two pairs of 1H double doublet at d 4.72–4.87, 4.62–4.77 are in agreement with amethine proton of tryptophan. Signals at 4.72–4.87 and 4.62– 4.77 in the COSY spectrum indicated methylenes adjacent to
Boc-Trp-OH+ NH2-Trp-OMe iii
Boc-Trp-Trp-AA-OBzl
i
ii
Boc-Trp-Trp-OMe
iv
Boc-Trp-Trp-OH
NH2 -Trp-Trp-AA-OBzl
Scheme 1. Preparation of NH2-Trp-Trp-AA-OBzl. Reagents and conditions: (i) NMM, DCC, HOBt; (ii) 2 M NaOH; (iii) NH2-AA-OBzl, NMM, DCC, HOBt; (iv) hydrogen chloride in ethyl acetate (4 mol/L).
HO
OH C
+
H
O O
O 1
O
O
O
O
O
O CO 2Et
3
2
HO
ii
HO
i
HO
O
O
iii
COTrp-Trp-AA-OBzl
CO2 H 5-17
4
Scheme 2. Synthesis of 7-hydroxy-2-oxo-2H-chromene-3-carboxylic acid-Trp-TrpAA-OBzl compounds. Reagents and conditions: (i) diethyl malonate and piperidine; (ii) HCl; (iii) NH2-Trp-Trp-AA-OBzl, NMM, DCC, HOBt.
methines. 1H NMR chemical shifts values at d 3.22–2.87 suggested the presence of methylene protons of tryptophan. 1H NMR spectroscopic signals at d 4.68–3.94 are in agreement with a-methine proton of the AA in NH2-Trp-Trp-AA-OBzl. The in vitro cytotoxicity of the coumarin derivatives synthesized above was evaluated in human lung adenocarcinoma cells (A549), chronic myeloid leukemia cells (K562), human liver carcinoma cells (HepG2), Human Glioblastoma cells (A172), and human hepatocellular carcinoma cells (Bel-7402) using MTT assay.23 Doxorubicin (adriamycin, ADM) was used as positive control. In brief, cells were exposed to 4–17 of concentrations ranging from
Table 1 Structures of coumarin derivatives synthesized Compound No.
Chemical structure HO
O
Compound No.
O
Chemical structure
HO
O
HO
O
O
4
3 CO2Et
HO
O
CO2H
O
5
O
6
COTrp-Trp-Asp(OBzl)-OBzl
COTrp-Trp-OBzl
HO
O
O
7
HO
O
HO
O
O
8
COTrp-Trp-Gly-OBzl
HO
COTrp-Trp-Phe-OBzl
O
O
9
O
10
COTrp-Trp-Leu-OBzl HO
O
COTrp-Trp-Ile-OBzl
O
11
HO
O
HO
O
HO
O
O
12
COTrp-Trp-Val-OBzl HO
O
COTrp-Trp-Ser-OBzl
O
13
O
14
COTrp-Trp-Tyr-OBzl
COTrp-Trp-Thr-OBzl
HO
O
O
15
O
16
COTrp-Trp-Pro-OBzl HO
O
COTrp-Trp-Trp-OBzl
O
17 COTrp-Trp-Glu(OBzl)-OBzl
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1 lM to 200 lM for 48 h and cell survival was then determined. As shown in Table 2, the IC50 values of ADM in HepG2, K562, A172, Bel-7402 and A549 cells were found to be 10.1, 8.9, 18.5, 11.7 and 4.3 lM, respectively. 4–17 exhibited varied activities in the five cell lines and they were found most effective in K562 cells and least effective in Bel-7402 cells. In K562 cells, 5, 7, 9, 14, 15 and 16 were found to be most effective with IC50 values of lower than 30 lM. The IC50 of 9 (IC50, 14.7 lM) is lower than that of 10 (IC50, >200.0 lM) suggesting that the methyl group at the b or c position of an amino acid residue cause changes in the cytotoxicity. Compared to 8 (IC50, 70.3 lM), 14 (IC50, 19.6 lM) exhibited a higher cytotoxicity suggesting that the introduction of the additional hydroxyl group in the phenylalanine increased the cytotoxicity. In A172 cells, 7, 9 and 10 showed potent cytotoxic activity with IC50 values of lower than 27.3 lM and Compound 7 demonstrated the significant cytotoxic activity with an IC50 value of 10.1 lM and was better than the positive control doxorubicin ADM (IC50, 18.5 lM). In HepG2 cells, 7 was found to be most effective with an IC50 value of 16.4 lM. In A549 cells, 8 and 9 were found to be most effective with IC50 values of lower than 29.2 lM. The coumarin derivatives were found to be generally less potent in Bel-7402 cells. Among all the compounds 6 was found to be the most potent compounds of this series with an IC50 value of 45.5 lM, 5 and 9 displayed the prominent and broader spectrum of cytotoxic activities against all the five human tumor cell lines tested with four IC50 values lower than 49.2 lM and 22.9 lM, respectively. The results seem to suggest that the amino acid in the Trp-Trp-AA-OBzl at position 3 of coumarin ring plays an important role in the cytotoxicity observed. The anti-tumor activity of coumarin derivatives synthesized (4– 17) was evaluated in mice bearing S180.24 The mice were given a daily ip injection of 1 lmol/kg of 4–17 in 0.2 mL of normal saline (NS) for seven consecutive days with NS as the negative control. ADM at 1 lmol/kg (0.2 mL) dissolved in 0.9% saline was used as positive control. The tumor weights were found to be 1.05–0.56 g compared to 1.45 g in the negative control group. The tumor inhibition % in describing the anti-tumor effects was determined as:
Tumor Inhibition% ¼ ðC TÞ=C 100 T: average tumor weight of treated group; C: average tumor weight of negative control group The anti-tumor activity results of these compounds (4–17) are summarized in Table 3. The tumor inhibition % of the compounds tested were found to range from 27.3% (17) to 61.5% (14). Comparison of inhibition ratio values of Compound 4 (34.2%) and Compound 5 (48.0%) showed that the presence of Trp-Trp dipeptide
Table 2 IC50 values of 4–17 in K562, A172, BL-7402, A549 and HepG2 cell lines
a
Compoundsa
HepG2
K562
A172
Bel-7402
A549
4 5 6 7 8 9 10 11 12 13 14 15 16 17 ADM
>200.0 33.1 ± 0.9 72.4 ± 1.2 16.4 ± 0.5 >200.0 22.9 ± 0.7 104.5 ± 1.7 >200.0 76.7 ± 1.4 33.5 ± 0.6 59.4 ± 0.9 >200.0 >200.0 >200.0 10.1 ± 1.2
>200.0 29.1 ± 1.3 >200.0 23.9 ± 0.9 70.3 ± 1.4 14.7 ± 1.3 >200.0 >200.0 >200.0 32.9 ± 1.0 19.6 ± 1.1 27.6 ± 1.7 28.0 ± 0.9 >200.0 8.9 ± 0.3
>200.0 99.5 ± 1.0 67.8 ± 1.3 10.1 ± 0.6 147.5 ± 2.9 21.9 ± 0.6 26.0 ± 1.1 58.1 ± 0.9 165.7 ± 1.6 63.4 ± 0.9 35.4 ± 0.4 90.7 ± 0.9 >200.0 >200.0 18.5 ± 0.7
>200.0 49.2 ± 1.4 45.5 ± 0.9 100.0 ± 1.4 >200.0 94.9 ± 1.0 95.5 ± 1.6 49.1 ± 0.5 >200.0 98.1 ± 1.3 >200.0 >200.0 >200.0 >200.0 11.7 ± 0.7
>200.0 45.9 ± 0.6 >200.0 >200.0 29.2 ± 1.2 22.0 ± 0.9 49.1 ± 2.1 194.2 ± 0.9 >200.0 >200.0 71.0 ± 0.9 >200.0 >200.0 >200.0 4.3 ± 0.4
IC50 value is expressed by X SD lM and n = 6.
Table 3 Tumor inhibition % by 4–17 in S180 bearing mice Compounda
Dose (lmol/kg)
Tumor weight (g)
Tumor inhibition %
NS ADM 4 5 6 7 8 9 10 11 12 13 14 15 16 17
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
1.45 ± 0.29 0.41 ± 0.08 0.95 ± 0.10b 0.75 ± 0.07b 0.74 ± 0.14b 0.59 ± 0.12b 0.73 ± 0.13b 0.65 ± 0.09b 0.77 ± 0.10b 0.75 ± 0.13b 0.84 ± 0.13b 0.93 ± 0.15b 0.56 ± 0.11b 0.77 ± 0.14b 0.72 ± 0.11b 1.05 ± 0.19c
71.7 ± 5.6 34.2 ± 7.1 48.0 ± 7.7 49.0 ± 9.6 59.5 ± 8.8 50.0 ± 9.0 55.1 ± 7.4 46.8 ± 6.9 48.4 ± 9.5 42.2 ± 8.2 35.9 ± 5.0 61.5 ± 8.3 47.2 ± 9.7 50.4 ± 0.9 27.3 ± 4.2
a ADM = Positive control, NS = Vehicle, n = 12, tumor weight is expressed by X SD g. b Compared to NS p < 0.01. c Compared to NS p < 0.05.
can increased the activity, suggesting that the Trp-Trp dipeptide structure had some contribution to the activity. Compounds 7 and 14 showed the highest tumor inhibition (59.5% and 61.5%, respectively) in mice bearing S180. Compared to 8 (50.0%), 14 had prominent anti-tumor activities (61.5%). It implied that the free hydroxyl group of tyrosine might play a vital role in the anti-tumor activity. Compared to 9 (55.1%), 10 and 11 exhibited a lower activity (46.8% and 48.4%, respectively) suggesting that the methyl group at the b position and the isopropyl group at the a position of an amino acid residue is likely responsible for the reduced activity observed. Compound 7 without substituent at position-a exhibited potent anti-tumor activity (59.5%). Since the coumarin derivatives showed higher degree of tumor inhibition, the preliminary toxicity of 4–17 were evaluated during the administration. All the tested compounds caused neither obvious neurotoxic reaction including tremor, twitch, jumping, tetanus and supination nor death at tested dosage. Twenty-four hours after the last administration the mice were decapitated. Necropsy of the dead mice was carried out. The necropsy revealed no apparent changes in any organs. Brain, heart, liver, spleen and kidney were weighed. The spleen indexes were measured. Most of the tested compounds exhibited a relatively minor toxic effect or better than the positive control, ADM. The data are listed in Table 4. Besides, 7 was examined for the LD50. The results indicate that even the dose of 7 was up to 500 mg/kg the mice occurred no death. These suggest that 7 is comparatively non-toxic and its LD50 values should be more than 500 mg/kg. Since two structural elements coumarin and Trp-Trp of the coumarin derivatives synthesized, each has a planar polycyclic aromatic pharmacophore capable of stacking between DNA pairs at the intercalation sites,25 the intercalation of the coumarin derivatives with calf thymus (CT) DNA was investigated by UV absorption, fluorescence and circular dichroism (CD) spectroscopy using 7. The UV spectrum of CT DNA in PBS (pH 7.4, 160 lM) was recorded on a Shimadzu 2550 spectrophotometer from 200 to 350 nm. 2 mL of CT DNA in PBS was then titrated with 20 lL of Compound 7 in PBS (pH 7.4) solutions of 0, 2.0, 4.0, 6.0, 8.0 and 10.0 lM, respectively. As shown in Figure 1, the UV absorption of CT DNA gradually decreased from 1.052 to 0.974 with the increase of 7 concentration and a hypochromic effect (7.4%) was induced.26 The fluorescence spectrum of 7 in PBS (pH 7.4, 14 lM) was obtained and compared with that in the presence of CT DNA in PBS (final concentrations: 0, 1.5, 3.0, 4.5, 6.0 and 7.5 lM, respectively,
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Table 4 Effect of 4–17 on the organs weight and spleen index of S180 mice
a b c
Compounda
Brain weight (g)
Heart weight (g)
Liver weight (g)
Kidney weight (g)
Spleen index
NS ADM 4 5 6 7 8 9 10 11 12 13 14 15 16 17
0.30 ± 0.02 0.29 ± 0.02 0.29 ± 0.04 0.28 ± 0.03 0.31 ± 0.04 0.29 ± 0.03 0.27 ± 0.05 0.28 ± 0.05 0.24 ± 0.07b 0.26 ± 0.06 0.26 ± 0.05 0.29 ± 0.04 0.28 ± 0.05 0.24 ± 0.06b 0.25 ± 0.05b 0.26 ± 0.05c
0.12 ± 0.02 0.11 ± 0.01 0.12 ± 0.01 0.10 ± 0.01 0.13 ± 0.02 0.11 ± 0.02 0.11 ± 0.02 0.11 ± 0.01 0.10 ± 0.01 0.09 ± 0.01b 0.12 ± 0.02 0.12 ± 0.02 0.10 ± 0.02 0.09 ± 0.02b 0.12 ± 0.03 0.11 ± 0.01
1.84 ± 0.32 1.60 ± 0.31c 1.81 ± 0.40 1.73 ± 0.33 1.61 ± 0.26c 1.86 ± 0.44 1.78 ± 0.44 1.60 ± 0.39c 1.78 ± 0.34 1.78 ± 0.31 1.90 ± 0.36 1.82 ± 0.41 1.76 ± 0.26 1.87 ± 0.32 1.80 ± 0.35 1.85 ± 0.32
0.16 ± 0.04 0.16 ± 0.04 0.20 ± 0.02 0.14 ± 0.04 0.16 ± 0.04 0.20 ± 0.04 0.17 ± 0.04 0.18 ± 0.04 0.17 ± 0.04 0.16 ± 0.03 0.18 ± 0.04 0.17 ± 0.03 0.15 ± 0.05 0.14 ± 0.03 0.17 ± 0.04 0.18 ± 0.03
7.25 ± 1.60 5.76 ± 0.99b 8.44 ± 1.93 7.72 ± 1.72 5.92 ± 1.52b 7.92 ± 1.96 8.69 ± 2.12 5.41 ± 1.32b 7.94 ± 1.58 6.97 ± 1.44 8.15 ± 1.97 7.43 ± 1.57 7.12 ± 1.55 4.67 ± 1.04b 6.93 ± 1.36 7.34 ± 1.40
Dose of ADM and 4–17: 1 lmol/kg, ADM = Positive control, NS = Vehicle, n = 12, spleen index is expressed by X SD mg/kg body weight. Compared to NS p < 0.01. Compared to NS p < 0.05.
Figure 1. UV spectra of CT DNA (pH = 7.4, concentration 160 lM) in the absence and presence of 7 of 0, 2.0, 4.0, 6.0, 8.0 and 10.0 lM, respectively.
Figure 3. CD spectra of CT DNA (pH = 7.4, concentration 160 lM) in the absence and presence of 7 (concentration 100 lM).
Figure 2. Fluorescence spectra of 7 (concentration 14 lM) in the absence and presence of CT DNA (pH = 7.4) of 0, 1.5, 3.0, 4.5, 6.0 and 7.5 lM, respectively.
pH 7.4) on a Shimadzu RF-5310PC spectrofluorometer (excitation wavelength 401 nm and emission wave-length 447 nm).27 When the concentration of CT DNA was increased to 7.5 lM the fluorescence intensity of Compound 7 was decreased by 15.9% from 536.7 to 451.2 as shown in Figure 2. In our CD experiments a solution of CT DNA alone in PBS buffer and a solution of CT DNA plus the representative 7 in PBS buffer were incubated at 37 °C for 24 h, and then their CD spectra were obtained in Figure 3.28 The results revealed the effect of intercalation of 7 into base stacking. This study provided some useful information to understand the interaction mechanism at the molecular level. In summary, 7-hydroxycoumarin derivatives bearing Trp-TrpAA-OBzl at Position 3 were designed, synthesized and evaluated. In vitro cytotoxicity assays against five human carcinoma cell lines (K562, A172, BL-7402, A549 and HepG2) explored the cell selective anti-proliferation for individual compounds. The in vivo anti-tumor activity assays with S180 mice revealed 7 and 14 having the highest efficacy. The tumor inhibition % of 7 and 14 is similar to the positive control doxorubicin ADM. The UV and fluorescence spectra, as well as and circular dichroism spectroscopy of 7 with or without CT DNA suggest that the binding of the coumarin derivatives synthesized to
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DNA was intercalative. These compounds may be considered as a structural clue for the further studies. Acknowledgments This work was supported by the Academic Human Resources Development in Institutions of Higher Learning Under the Jurisdiction of Beijing Municipality (PHR201007114), the National Basic Research Program of China (2011CB504100), the National Natural Scientific Foundation of China (81030062) and Key National Science & Technology Specific Projects (2011ZX09102-003-01), Beijing Pharmacological Society (2013BI-01). Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.09. 051. References and notes 1. Jimenez-Orozco, F. A.; Molina-Guarneros, J. A.; Mendoza-Patino, N.; LeonCedeno, F.; Flores-Perez, B.; Santos-Santos, E.; Mandoki, J. J. Melanoma Res. 1999, 9, 243. 2. Laurin, P.; Ferroud, D.; Klich, M.; Dupis-Hamelin, C.; Mauvais, P.; Lassaigne, P.; Bonnefoy, A.; Musicki, B. Bioorg. Med. Chem. Lett. 1999, 9, 2079. 3. Hoult, J. R. S.; Paya, M. Gen. Pharmacol. 1996, 27, 713. 4. Pillai, S. P.; Menon, S. R.; Mitscher, L. A.; Pillai, C. A.; Shankel, D. M. J. Nat. Prod. 1999, 62, 1358. 5. Kimura, Y.; Okuda, H.; Arichi, S.; Baba, K.; Kozawa, M. Biochim. Biophys. Acta 1985, 834, 224.
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