European Journal of Medicinal Chemistry 95 (2015) 166e173
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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Design and synthesis of celastrol derivatives as anticancer agents Wen-Jian Tang a, 1, Jing Wang a, 1, Xu Tong a, Jing-Bo Shi a, Xin-Hua Liu a, b, *, Jun Li a, ** a b
School of Pharmacy, Anhui Medical University, Hefei, 230032, PR China State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 January 2015 Received in revised form 16 March 2015 Accepted 17 March 2015 Available online 18 March 2015
A series of celastrol derivatives as potential telomerase inhibitors were designed and synthesized. The bioassays demonstrated that title compounds displayed potent anticancer activities against SGC-7901, SMMC-7721, MGC-803 and HepG-2 cell lines, among them, compounds 3c and 3d which containing hydrophilicity moieties exhibited high anti-proliferative activities (IC50 ¼ 0.10e1.22 mM). The preliminary mechanism of antitumor action indicated that title compound 3c could induce significant SMMC-7721 cells apoptosis. A modified TRAP assay showed that compounds 3c and 3d displayed the most potent inhibitory activity with IC50 values at 0.11 and 0.34 mM, respectively. And there was a good correlation between telomerase inhibition and anti-proliferative inhibition of SMMC-7721 cells. Moreover, molecular docking indicated that the active compound 3c was nicely bound into the telomerase hTERT active site, hydrophobic, van der Waals and two hydrogen bond interactions with conserved residues ASP 628 and TYR 949 were found. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Celastrol derivatives Anticancer activity Telomerase Cell apoptosis
1. Introduction Telomerase plays a pivotal role in bypassing cell senescence and maintaining telomere homeostasis. Telomerase reverse transcriptase (TERT) is a catalytic subunit of the enzyme telomerase [1e3]. TERT has been reported to be over-expressed in more than 90% of cancer cells, while most normal tissues and cells contain inactivated telomerase, thereby, telomerase plays a critical role in sustained proliferation and survival potentials of various cancer cells [4,5]. Numerous evidences have suggested that TERT could modulate the expression of numerous genes including cell cycle regulation and cellular signaling [6,7]. Therefore, targeting TERT is an important strategy for the development of cancer agents [8,9]. It was of our interest to utilize rational chemical approaches and discover novel natural compounds with anticancer as telomerase inhibitors from Traditional Chinese Medicine (TCM). Focused on telomerase TERT (pdb: 3DU6), we disclosed the X-ray crystal structure of some molecule inhibitors bound to active site of TERT [6,10,11], based on this, in our previous study, a compound 18aGAMG (pentacyclic triterpene scaffold, Fig. 1A) inhibited the
* Corresponding author. School of Pharmacy, Anhui Medical University, Hefei, Anhui 230032, PR China. ** Corresponding author. E-mail addresses: [email protected] (X.-H. Liu), [email protected] (J. Li). 1 W.J.T and J.W. contributed equally to this work. http://dx.doi.org/10.1016/j.ejmech.2015.03.039 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
expression of TERT and with selectivity activity against tumor cells versus human somatic cells was discovered [12]. But, unfortunately, the activity of this compound against to tumor cell lines is not high enough, herein, in continuation to extend our research on telomerase inhibitors, we began to look for other types of pentacyclic triterpene scaffolds according to above findings. Among these compounds, celastrol (Fig. 1B) is an active compound isolated from the root extracts of TCM Tripterygium wilfordii (Thunder of God Vine). Celastrol is a pentacyclic triterpenoid and belongs in the family of quinone methides. Celastrol has been investigated widely for its anti-inflammatory and anticancer activity [13,14]. This triterpene modulates multiple molecular targets such as Hsp90-Cdc37, TNF-a, NF-kB, VEGF, Akt, CXCR4, proinflammatory cytokines and chemokines involved playing a major role in all three steps (initiation, proliferation and progression) of carcinogenesis [15,16]. So far, celastrol has been shown to have beneficial effects on a variety of cancers in vitro and in vivo, including pancreatic cancer, hepatocellular carcinomas, squamous cancer, and prostate cancer [17e20], suggesting it might be developed as a potential cancer treatment. Although celastrol has important pharmacological activity, its poor water solubility and high toxicity restricts its application. Thus, some structure modifications were carried out to improve solubility or reduce toxicity, which focus on either the esterification or amidification of 20-carboxylic acid or the reduction products of A/B rings of celastrol [21e26]. Structural modifications at the C-2, 3
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O HO H
O
COOH HOOC HO HO
H O O OH
H
A: 18α-GAMG
H
Extract
COOH HO
O
H
HO
B: Celastrol
Title Pentacyclic triterpene scaffold
Fig. 1. The general design strategy in this study.
positions were reported as the inducers of heat shock response, while those at C-6 position displayed better anticancer activity. However, previous studies also suggested that the intact quinone methide moiety was crucial for its cytotoxic activity in cancer cell lines and neuroprotective effect [21,23]. In this study, based on improving celastrol water solubility and to further understand the preliminary structureeactivity relationship (SAR) against cancer cells, we rationally designed and synthesized a series of celastrol derivatives with the intact quinone methide group. The anti-proliferative activity and telomerase inhibitive activity of these derivatives were evaluated. 2. Results and discussion 2.1. Chemistry Carboxylic acid can be transferred to ether by alkylation reaction with the catalysis of alkali. Compound 2 was obtained by the esterification at 20-carboxylic acid of celastrol (1) in the presence of iodoethane and catalyst NaHCO3 in anhydrous DMF with 72% yield. Amides are commonly formed via reactions of an “activated” carboxylic acid with an amine. Compounds 3a~3i were obtained from amide condensation reaction catalyzed by EDC∙HCl, HOBt and TEA in anhydrous CH2Cl2 conditions with 26%~56% yield (Scheme 1). The products were purified by extraction, column chromatography and recrystallization, and their structures were confirmed by 1H NMR, 13C NMR and HRMS according to the literature [27]. Purify was evaluated under HPLC conditions and exceeded 96%. 2.2. Crystal structure analysis The structure of compound 3a was determined by X-ray crystallography. Crystal data of 3a: Clear light orange crystals obtained from EtOH/H2O, yield, 77%; mp 133e135 C; C30H41NO3, M ¼ 463.64, Monoclinic, space group P21; a ¼ 11.6031(5), b ¼ 8.1736(2), c ¼ 13.9701(7) (Å); a ¼ 90, b ¼ 106.252(5), g ¼ 90, V ¼ 1271.96(10) Å3, T ¼ 294 K, Z ¼ 2, Dc ¼ 1.211 g/cm3, F(000) ¼ 504, Reflections collected/Independent reflections ¼ 4621/3696, Data/ restraints/parameters ¼ 4621/1/315, Goodness of fit on F2 ¼ 1.045, Fine, R1 ¼ 0.0480, wR(F2) ¼ 0.1248. The molecular structure of compound 3a was shown in Fig. 2.
The absolute configuration of compound 3a was determined using CuKa radiation (l ¼ 1.54184 Å), that is C5(S), C11(S), C15(R), C17(S), C20(R) and C27(S). In compound 3a, rings A and B showed plane conformations, while the other rings showed mixed forms between envelope and half-chair conformations with atoms in rings C, D and E, respectively. Rings D and E were cis-form, which was similar to that of 18b-GAMG [12]. Crystallographic data (excluding structure factors) for the structure had been deposited with the Cambridge Crystallographic Data Center as supplementary publication No. CCDC 1034724 [28]. 2.3. In vitro anticancer activity All title compounds were evaluated for their anticancer activities in vitro against SGC-7901 (human gastric cancer cells), SMMC7721 (human hepatoma cells), MGC-803 (human gastric cancer cells) and HepG-2 (human hepatoma cells) cell lines, also included the activity of references 5-Fluorouracil and doxorubicin (AMD). The cells were allowed to proliferate in presence of tested material for 48 h, and the results are reported in terms of IC50 values (Table 1). Celastrol and its derivatives showed remarkable antiproliferative effects. Among them, compounds 1, 3c, 3d and 3i displayed potent inhibitory activities (IC50 1 mM, as 1, 3c, 3d, IC50 ¼ 0.15, 0.16, 0.10 mM for SGC-7901 cells; as 1, 3c, 3d, IC50 ¼ 0.58, 0.30, 0.61 mM for SMMC-7721 cells; as 3c, 3i, IC50 ¼ 0.39, 1.00 mM for MGC-803 cells; as 3c, 3d, IC50 ¼ 0.61, 0.28 mM for HepG-2 cells, respectively), which were more potent than the positive control 5Fluorouracil and AMD. Besides, compound 3h showed potent anticancer activities against SGC-7901 and SMMC-7721 cell lines, surpassing that of the positive control 5-Fluorouracil. Subsequently preliminary SAR studies were performed to determine how the substituent at 20-carboxylic acid affected the anticancer activity. Celastrol without any substituent group also showed good anti-proliferative activity. Firstly, the esterification of 20-carboxylic acid to form compound 2 showed lowest anticancer activities, whose IC50 values (9.28e15.19 mM) was lower than those of other compounds and the positive control. Secondly, the amidification of 20-carboxylic acid to form compounds 3a~3i displayed different anticancer activity (IC50 ¼ 0.10e19.97 mM). Compounds 3c, 3d with b-hydroxyl-ethylamide substituent exhibited potent anticancer activity against all four cell lines, while compounds 3h,
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O
O OH
R
A or B
H
O
H
O
HO
HO
2, 3
1
2: R = -OCH2CH3; 3a: R = -NHCH3; 3b: R = -N(CH3)2; 3c: R = -NHCH2CH2OH 3d: R = -NH-i-Propanol; 3e: R = -NH(CH2)3CH3; 3f: R =
N
O
; 3g: R =
N
N
N
O
; 3h: R =
N
N
OH
; 3i: R =
N
N
N
Reagents and conditions: (A) NaHCO3, DMF, iodoethane, rt; (B) EDC·HCl, HOBt, TEA, CH2Cl2. Scheme 1. Synthesis of title compounds 2 and 3.
Table 1 In vitro anticancer activity of the synthesized compounds against SGC-7901, SMMC7721, MGC-803 and HepG2 cell lines.a Compound
IC50 (mM)b SGC-7901
Fig. 2. ORTEP drawing of compound 3a.
3i showed moderate anticancer activity. Finally, compared to celastrol, compounds 3c, 3d increased 4.0, 1.3 and 6.6, 14.3 times anti-proliferative activity for MGC-803 and HepG-2 cells, respectively.
1 2 3a 3b 3c 3d 3e 3f 3g 3h 3i 5- Fluorouracilc AMDc
0.15 11.50 9.38 10.50 0.16 0.10 4.90 8.74 8.10 3.81 1.33 6.12 1.45
± ± ± ± ± ± ± ± ± ± ± ± ±
0.07 2.44 2.11 1.91 0.02 0.02 0.45 1.19 2.01 0.51 0.41 0.89 0.09
SMMC-7721 0.58 9.28 7.62 19.97 0.30 0.61 6.88 10.70 12.32 1.84 2.58 4.87 1.22
± ± ± ± ± ± ± ± ± ± ± ± ±
0.18 0.99 1.61 1.66 0.06 0.07 0.91 2.28 2.27 0.51 0.54 0.27 0.12
MGC-803 1.55 12.00 24.10 6.30 0.39 1.22 3.55 30.70 13.40 10.30 1.00 3.58 1.19
± ± ± ± ± ± ± ± ± ± ± ± ±
0.25 2.10 2.70 0.98 0.01 0.08 0.87 4.11 1.33 2.10 0.17 0.40 0.07
HepG2 4.01 15.19 10.60 11.20 0.61 0.28 3.07 22.70 16.38 8.70 5.21 5.86 0.87
± ± ± ± ± ± ± ± ± ± ± ± ±
1.21 1.21 2.45 0.98 0.11 0.07 0.51 4.87 2.15 2.12 0.44 0.73 0.10
Negative control DMSO, no activity. a The data represented the mean of three experiments in triplicate and were expressed as means ± SD; only descriptive statistics were done in the text. b The IC50 value was defined as the concentration at which 50% survival of cells was observed. The results are listed in the table. c Used as a positive control.
2.5. Telomerase activity assay 2.4. Induction of apoptosis by compound 3c The annexin V-FITC/PI apoptosis detection kit was used to determine whether compound 3c meditated inhibition of growth and proliferation was associated with apoptosis. As shown in Fig. 3, only a small percentage of untreated SMMC-7721 (2.0%) cells bound with annexin V-FITC. In contrast, the percentage of annexin V-FITC binding SMMC-7721 cells significantly increased after treatment with 0.5 mM compound 3c (2.0%e61.2%, p < 0.05). In a word, data points were dispersed and shifted to the Q3 side when SMMC-7721 cells treated with compound 3c, indicating that the cells moved to the early apoptotic stage. The experimental results demonstrated that compound 3c induced apoptosis of SMMC-7721 cells.
The synthesized compounds were assayed for telomerase inhibition using an MGC-803 cell extract, and BIBR1532 was selected as the reference compound. The results were presented as mean ± SD. As shown in Table 2, the tested compounds displayed good telomerase inhibitory. Among them, compounds 3c and 3d displayed strong telomerase inhibitory activity with IC50 values of 0.11 and 0.34 mM respectively, which was superior to that of the positive control BIBR1532. Then, an analysis between telomerase inhibition and inhibition of SMMC-7721 cellular proliferation indicated that there was a good correlation with an R value of 0.968, as evidenced in Fig. 4. This demonstrated that the potent inhibitory effects of title compounds on the cell proliferation assay were causally related to
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Fig. 3. Compound 3c induced apoptosis in SMMC-7721 using annexin V-FITC/PI. (A)e(B) Treatment with 0 and 0.5 mM compound 3c for 48 h, respectively. The experiments were repeated three times and representative photographs are shown. A: normal SMMC-7721, B: SMMC-7721þ0.5 mM compound 3c. Q1: necrotic cells, Q2: viable apoptotic cells, Q3: non-viable apoptotic cells, Q4: normal cells.
further optimize the structure of celastrol with anticancer activity as potential telomerase inhibitors.
Table 2 LogP and inhibitory effects of title compounds against telomerase. Compound
logP
IC50 (mM) telomerasea
1 2 3a 3b 3c 3d 3f 3g 3h 3i BIBR1532b
4.38 4.98 3.96 4.20 3.44 3.49 3.80 3.50 3.44 3.95
0.78 18.55 15.10 21.01 0.11 0.34 8.01 15.17 2.01 1.41 0.41
a b
± ± ± ± ± ± ± ± ± ± ±
0.17 1.42 1.61 1.95 0.02 0.07 0.81 1.80 0.33 0.20 0.05
Telomerase supercoiling activity. BIBR1532 is reported as a control.
2.6. The octanol/water partition coefficient (log P) In order to know whether is the polarity of celastrol derivative affects its anti-proliferative activity in this study. Theoretical octanol/water partition coefficient (log P) of synthesized compounds was measured by chemical software ChemDraw 12.0. As showed in Table 2, the inhibitory activity of synthesized compounds was associated with log P value. Compared to celastrol (1, log P ¼ 4.38), compounds 3c and 3d with b-hydroxyl-ethylamide moiety showed better hydrophilic (log P ¼ 3.44 and 3.59, respectively), which displayed higher anticancer activity. 2.7. Molecular docking
their telomerase inhibitory activities. These results suggested that compounds 3c and 3d were excellent anticancer agents with potent telomerase inhibitory, which also pointed out the direction for us to
A three-dimension human telomerase model [29] and an advanced docking method e IFD (Induced Fit Docking) of €dinger were employed to explore the binding mode of Schro BIBR1532, a potent hTERT inhibitor. The docked complex hTERTeBIBR1532 was then done a 10 ns MD simulation. Compound 3c was docked into the active site of the modeled hTERTeBIBR1532 complex to see its putative binding pose. As shown in Fig. 5, a typical docking pose demonstrates that the flexible side chain of compound 3c forms two hydrogen bonds with the receptor: one is between the hydroxyl group and residue of ASP 628; another is between the hydroxyl group and residue of TYR 949. The hydrophobic and rigid fused ring of compound 3c, whereas, is inserted into the active site of hTERT and has hydrophobic and van der Waals interactions with the receptor. 3. Conclusion
Fig. 4. The linear regression results of cell lines versus kinases to explore the correlation between pIC50's for the inhibition of telomerase and SMMC-7721 cellular proliferation, R ¼ 0.968.
In conclusion, based on our previous study, focused on pentacyclic triterpene moiety, a series of celastrol derivatives as potential telomerase inhibitors were designed and synthesized. In vitro bioassays demonstrated that celastrol derivatives displayed potent anticancer activities against SGC-7901, SMMC-7721, MGC-803 and HepG-2 cell lines, among them, compounds 3c and 3d exhibited high anti-proliferative activities (IC50 ¼ 0.10e1.22 mM), which hydrophilicity might be one of the reasons for high anticancer activity. The telomerase assay showed that there was a good correlation
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CDCl3), d (ppm): 6.99 (d, 1H, J ¼ 4.7 Hz, H-6), 6.50 (s, 1H, H-1), 6.32 (d, 1H, J ¼ 4.8 Hz, H-7), 3.94 (m, 2H, OCH2CH3), 2.46 (d, 1H, J ¼ 10.5 Hz, H-19a), 2.18 (s, 3H, CH3-23), 1.42 (s, 3H, CH3-25), 1.23 (s, 3H, CH3-26), 1.17 (t, 3H, J ¼ 4.8 Hz, OCH2CH3), 1.14 (s, 3H, CH3-30), 1.07 (s, 3H, CH3-28), 0.53 (s, 3H, CH3-27); 13C NMR (100 MHz, CDCl3), d (ppm): 10.5 (C-23), 14.2 (OCH2CH3), 18.3 (C-27), 21.8 (C26), 28.8 (C-15), 29.8 (C-12), 30.0 (C-30), 30.8 (C-17), 30.9 (C-21), 31.8 (C-19), 33.0 (C-28), 33.7 (C-11), 34.9 (C-22), 36.5 (C-16), 38.4 (C-25), 39.6 (C-13), 40.4 (C-20), 43.1 (C-9), 44.4 (C-18), 45.2 (C-14), 60.5 (OCH2CH3), 117.4 (C-4), 118.3 (C-7), 119.7 (C-1), 127.6 (C-5), 134.4 (C-6), 146.2 (C-3), 164.9 (C-10), 170.4 (C-8), 178.3, 178.6 (2 C]O). HR-ESI-MS: m/z [MþH]þ calcd for C31H43O4: 479.3156; found: 479.3157; HPLC: tR 4.40 min, PHPLC 99.7%. 4.3. General procedure for the synthesis of title compound 3 € dinger. Fig. 5. Binding mode of compound 3c to hTERT yielded by Glide 5.9 of Schro
between telomerase inhibition and anti-proliferative inhibition of SMMC-7721 cells, and compounds 3c and 3d displayed the most potent inhibitory activity with IC50 values at 0.11 and 0.34 mM respectively. Molecular docking indicated that the active compound 3c was nicely bound to telomerase hTERT via two hydrogen bonds and van der Waals interactions. Moreover, the mechanism of antitumor action indicated that title compound 3c could induce significant cell apoptosis in SMMC-7721 cells. These results support further studies to optimize structure of celastrol as telomerase inhibitors in the future. 4. Experimental section 4.1. Chemistry All reagents were purchased from commercial sources and were used without further purification. Melting points (uncorrected) were determined on a XT4MP apparatus (Taike Corp., Beijing, China). 1H NMR and 13C NMR spectra were recorded on Bruker AV400 or AV-300 MHz instruments in CDCl3 using tetramethylsilane (TMS) as the internal standard. Chemical shifts are reported in ppm (d). Coupling constants are reported in hertz. The multiplicity is defined by s (singlet), d (doublet), t (triplet), or m (multiplet). Mass spectra were recorded on a LTQ-Orbitrap XL (Thermo-Fisher Scientific, LLC). Column and thin-layer chromatography (CC and TLC, resp.) were performed on silica gel (200e300 mesh) and silica gel GF254 (Qingdao Marine Chemical Factory) respectively. HPLC was performed on a Shimadzu LC-20AT system equipped with an autoinjector and a UV detector set at 254 nm. Shimadzu software was used to calculate peak areas. Compounds were separated on a Spherisorb C18 column from DEAIC (Dalian, China). The mobile phase was a mixture of MeOH/H2O (95:5 / 85:15) for compounds 3a-3i. HPLC retention times (HPLC tR) were obtained at a flow rate of 1.0 mL/min. 4.2. The synthesis of title compound 2 To a DMF (2 mL) solution of celastrol (45 mg, 0.10 mmol) was added NaHCO3 (19 mg, 0.22 mmol) and iodoethane (18 mL, 0.22 mmol). The reaction mixture was stirred for 3 h at room temperature, then washed twice with water, dried with Na2SO4, filtered, and concentrated in vacuo. The residue was purified by crystallization from ethanol to give title compound 2. 4.2.1. 3-Hydroxy-9b,13a-dimethyl-2-oxo-24,25,26-trinoroleana1(10),3,5,7-tetraen- 29-oic acid ethyl ester Red powder, yield, 72%; mp 116e119 C; 1H NMR (400 MHz,
To a CH2Cl2 (5 mL) solution of celastrol (45 mg, 0.10 mmol) was added EDC∙HCl (43 mg, 0.22 mmol), HOBt (30 mg, 0.22 mmol), and corresponding amine (2e3 eq.), the mixture was stirred 30 min at 0 C. After TEA (50 mL) was added, the reaction solution was stirred overnight at room temperature. The mixture was washed twice with water, dried with Na2SO4, filtered, and concentrated in vacuo. The residue was purified by chromatography on a silica gel column (dichloromethane/methanol, 10:0 / 10:1) to give title compounds 3ae3i. 4.3.1. 3a: 3-Hydroxy-9b,13a-dimethyl-2-oxo-24,25,26trinoroleana-1(10),3,5,7-tetraen-29-oic amide, N-methyl Red powder, yield, 49%; mp 131e134 C; 1H NMR (400 MHz, CDCl3), d (ppm): 6.98 (d, 1H, J ¼ 4.4 Hz, H-6), 6.50 (s, 1H, H-1), 6.31 (d, 1H, J ¼ 4.8 Hz, H-7), 5.75 (m, 1H, J ¼ 2.8 Hz, NH), 2.64 (d, 3H, J ¼ 2.8 Hz, NCH3), 2.43 (d, 1H, J ¼ 10.4 Hz, H-19a), 2.18 (s, 3H, CH323), 1.41 (s, 3H, CH3-25), 1.23 (s, 3H, CH3-26), 1.12 (s, 3H, CH3-30), 1.09 (s, 3H, CH3-28), 0.59 (s, 3H, CH3-27); 13C NMR (100 MHz, CDCl3), d (ppm): 10.5 (C-23), 18.3 (C-27), 21.9 (C-26), 26.7 (NCH3), 28.8 (C-15), 29.6 (C-12), 29.9 (C-30), 30.3 (C-21), 31.1 (C-17), 31.5 (C19), 31.8 (C-28), 33.7 (C-11), 33.8 (C-28), 36.6 (C-16), 38.4 (C-25), 39.5 (C-13), 40.5 (C-20), 43.2 (C-9), 44.6 (C-18), 45.3 (C-14), 117.3 (C4), 118.2 (C-7), 119.7 (C-1), 127.6 (C-5), 134.2 (C-6), 146.2 (C-3), 165.0 (C-10), 170.5 (C-8), 178.5 (C]O), 178.6 (C]O). HR-ESI-MS: m/z [MþH]þ calcd for C30H42NO3: 464.3159; found: 464.3153; HPLC: tR 8.80 min, PHPLC 99.7%. 4.3.2. 3b: 3-Hydroxy-9b,13a-dimethyl-2-oxo-24,25,26trinoroleana-1(10),3,5,7-tetraen-29-oic amide, N,N-dimethyl Red powder, yield, 33%; mp 141e144 C; 1H NMR (400 MHz, CDCl3), d (ppm): 7.04 (d, 1H, J ¼ 6.8 Hz, H-6), 6.54 (s, 1H, H-1), 6.36 (d, 1H, J ¼ 6.8 Hz, H-7), 3.20 (s, 3H, NCH3), 2.81 (s, 3H, NCH3), 2.42 (d, 1H, J ¼ 16.0 Hz, H-19a), 2.35 (d, 1H, J ¼ 14.1 Hz, H-19b), 2.22 (s, 3H, CH3-23), 1.46 (s, 3H, CH3-25), 1.28 (s, 6H, CH3-26, CH3-30), 1.14 (s, 3H, CH3-28), 0.54 (s, 3H, CH3-27); 13C NMR (100 MHz, CDCl3), d (ppm): 10.3 (C-23), 18.5 (C-27), 22.1 (C-26), 28.8 (C-15), 29.7 (C12), 30.0 (C-30), 30.4 (C-21), 30.8 (C-17), 31.0 (C-19), 32.0 (C-28), 33.5 (C-11), 34.4 (C-22), 36.0 (2 NCH3), 36.4 (C-16), 38.3 (C-25), 39.6 (C-13), 40.2 (C-20), 42.9 (C-9), 44.9 (C-18), 45.1 (C-14), 117.3 (C4), 118.3 (C-7), 119.4 (C-1), 127.4 (C-5), 134.4 (C-6), 146.0 (C-3), 164.8 (C-10), 170.2 (C-8), 176.9 (C]O), 178.3 (C]O). HR-ESI-MS: m/z [MþH]þ calcd for C31H44NO3: 478.3316; found: 478.3315; HPLC: tR 11.34 min, PHPLC 99.7%. 4.3.3. 3c: 3-Hydroxy-9b,13a-dimethyl-2-oxo-24,25,26trinoroleana-1(10),3,5,7-tetraen-29-oic amide, N-(2-hydroxy)ethyl Red powder, yield, 38%; mp 182e185 C; 1H NMR (400 MHz, CDCl3), d (ppm): 6.98 (d, 1H, J ¼ 4.6 Hz, H-6), 6.49 (s, 1H, H-1), 6.30 (d, 1H, J ¼ 4.6 Hz, H-7), 6.29 (s, 1H, NH), 3.61 (m, 2H, CH2OH), 2.41
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(d, 1H, J ¼ 10.4 Hz, H-19a), 2.18 (s, 3H, CH3-23), 1.40 (s, 3H, CH3-25), 1.22 (s, 3H, CH3-26), 1.14 (s, 3H, CH3-30), 1.09 (s, 3H, CH3-28), 0.61 (s, 3H, CH3-27); 13C NMR (100 MHz, CDCl3), d (ppm): 10.5 (C-23), 18.5 (C-27), 21.9 (C-26), 28.8 (C-15), 29.6 (C-12), 29.9 (C-30), 30.3 (C-21), 31.0 (C-17), 31.3 (C-19), 31.8 (C-28), 33.7 (C-11), 34.1 (C-22), 36.5 (C16), 38.4 (C-25), 39.5 (C-13), 40.5 (C-20), 42.7 (NHCH2), 43.2 (C-9), 44.5 (C-18), 45.2 (C-14), 62.2(CH2OH), 117.3 (C-4), 118.2 (C-7), 119.7 (C-1), 127.6 (C-5), 134.2 (C-6), 146.2 (C-3), 165.0 (C-10), 170.5 (C-8), 178.5 (C]O), 178.6 (C]O). HR-ESI-MS: m/z [MþH]þ calcd for C31H44NO4: 494.3265; found: 494.3266; HPLC: tR 6.60 min, PHPLC 99.4%. 4.3.4. 3d: 3-Hydroxy-9b,13a-dimethyl-2-oxo-24,25,26trinoroleana-1(10),3,5,7-tetraen-29-oic amide, N-(2-hydroxy) propyl Red powder, yield, 33%; mp 90e92 C; 1H NMR (300 MHz, CDCl3), d (ppm): 7.02 (d, 1H, J ¼ 6.9 Hz, H-6), 6.98 (s, 1H, OH), 6.53 (s, 1H, H-1), 6.34 (d, 1H, J ¼ 7.2 Hz, H-7), 5.85 (d, 1H, J ¼ 6.9 Hz, NH), 3.50 (m, 2H, CH2OH), 2.40 (d, 1H, J ¼ 15.8 Hz, H-19a), 2.21 (s, 3H, CH3-23), 1.45 (s, 3H, CH3-25), 1.26 (s, 3H, CH3-26), 1.25 (d, 3H, J ¼ 3.0 Hz, CHCH3), 1.16 (s, 3H, CH3-30), 1.11 (s, 3H, CH3-28), 0.67 (s, 3H, CH3-27). 13C NMR (75 MHz, CDCl3), d (ppm): 10.5 (C-23), 18.3 (CHCH3), 18.7 (C-27), 22.0 (C-26), 28.9 (C-15), 29.8 (C-12), 29.9 (C30), 30.4 (C-21), 31.0 (C-17), 31.8 (C-19), 32.1 (C-28), 33.7 (C-11), 34.2 (C-22), 36.5 (C-16), 38.4 (C-25), 39.6 (C-13), 40.5 (C-20), 43.2 (C-9), 44.6 (C-18), 45.2 (C-14), 47.9 (NHCH), 67.3 (CH2OH), 117.3 (C4), 118.3 (C-7), 119.8 (C-1), 127.6 (C-5), 134.3 (C-6), 146.2 (C-3), 164.9 (C-10), 170.2 (C-8), 178.6 (C]O), 178.7 (C]O). HR-ESI-MS: m/z [MþH]þ calcd for C32H46NO4: 508.3421; found: 508.3416; HPLC: tR 10.28 min, PHPLC 99.5%. 4.3.5. 3e: 3-Hydroxy-9b,13a-dimethyl-2-oxo-24,25,26trinoroleana-1(10),3,5,7-tetraen-29-oic amide, N-n-butyl Red powder, yield, 56%; mp 80e82 C; 1H NMR (300 MHz, CDCl3), d (ppm): 7.02 (d, 1H, J ¼ 6.3 Hz, H-6), 7.01 (s, 1H, OH), 6.53 (s, 1H, H-1), 6.35 (d, 1H, J ¼ 7.2 Hz, H-7), 5.73 (m, 1H, NH), 3.10 (m, 2H, CH2NH), 2.46 (d, 1H, J ¼ 14.3 Hz, H-19a), 2.21 (s, 3H, CH3-23), 1.44 (s, 3H, CH3-25), 1.26 (s, 3H, CH3-26), 1.15 (s, 3H, CH3-30), 1.12 (s, 3H, CH3-28), 0.86 (t, 3H, J ¼ 7.2 Hz, CH2CH3), 0.64 (s, 3H, CH3-27); 13C NMR (75 MHz, CDCl3), d (ppm): 10.4 (C-23), 14.0 (CH2CH3), 18.4 (C27), 20.4 (CH2CH3), 22.0 (C-26), 28.8 (C-15), 29.6 (C-12), 30.4 (C-30), 31.0 (C-21), 31.3 (C-17), 31.5 (CH2CH2CH3), 31.8 (C-19), 33.7 (C-28), 34.0 (C-11), 35.2 (C-22), 36.5 (C-16), 38.3 (C-25), 39.5, 39.6 (C-13, NHCH2), 40.4 (C-20), 43.2 (C-9), 44.5 (C-18), 45.2 (C-14), 117.2 (C-4), 118.2 (C-7), 119.7 (C-1), 127.6 (C-5), 134.3 (C-6), 146.2 (C-3), 165.0 (C10), 170.5 (C-8), 177.8 (C]O), 178.5 (C]O). HR-ESI-MS: m/z [MþH]þ calcd for C33H48NO3: 506.3629; found: 506.3613; HPLC: tR 15.80 min, PHPLC 99.7%. 4.3.6. 3f: 3-Hydroxy-9b,13a-dimethyl-2-oxo-24,25,26trinoroleana-1(10),3,5,7-tetraen-29-oic morpholine Red powder, yield, 37%; mp 133e137 C; 1H NMR (400 MHz, CDCl3), d (ppm): 6.99 (d, 1H, J ¼ 4.7 Hz, H-6), 6.50 (s, 1H, H-1), 6.32 (d, 1H, J ¼ 4.8 Hz, H-7), 3.61 (m, 8H, 2 NCH2, 2 OCH2), 2.28 (d, 1H, J ¼ 9.8 Hz, H-19a), 2.18 (s, 3H, CH3-23), 1.43 (s, 3H, CH3-25), 1.26 (s, 3H, CH3-26), 1.25 (s, 3H, CH3-30), 1.11 (s, 3H, CH3-28), 0.58 (s, 3H, CH3-27); 13C NMR (100 MHz, CDCl3), d (ppm): 10.5 (C-23), 18.9 (C27), 22.6 (C-26), 29.1 (C-15), 29.9 (C-12), 30.2 (C-30), 30.8 (C-21), 30.9 (C-17), 31.1 (C-19), 32.1 (C-28), 33.5 (C-11), 34.3 (C-22), 36.2 (2 NCH2), 36.4 (C-16), 38.5 (C-25), 39.8 (C-13), 40.3 (C-20), 43.1 (C-9), 44.9 (C-18), 45.0 (C-14), 66.6 (2 OCH2), 117.3 (C-4), 118.5 (C7), 119.7 (C-1), 127.6 (C-5), 134.3 (C-6), 146.2 (C-3), 164.7 (C-10), 170.1 (C-8), 176.4 (C]O), 178.5 (C]O). HR-ESI-MS: m/z [MþH]þ calcd for C33H46NO4: 520.3421; found: 520.3419; HPLC: tR 8.55 min, PHPLC 97.7%.
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4.3.7. 3g: 3-Hydroxy-9b,13a-dimethyl-2-oxo-24,25,26trinoroleana-1(10),3,5,7-tetraen-29-oic piperazine, N-(2morpholinyl)ethyl Red powder, yield, 26%; mp 118e122 C; 1H NMR (400 MHz, CDCl3), d (ppm): 6.99 (d, 1H, J ¼ 4.7 Hz, H-6), 6.50 (s, 1H, H-1), 6.32 (d, 1H, J ¼ 4.8 Hz, H-7), 3.68 (m, 8H, morpholine 4 CH2), 2.48e2.45 (m, 12H, piperazine 4 CH2, NCH2CH2N), 2.18 (s, 3H, CH3-23), 1.42 (s, 3H, CH3-25), 1.25 (s, 3H, CH3-26), 1.23 (s, 3H, CH3-30), 1.10 (s, 3H, CH3-28), 0.56 (s, 3H, CH3-27); 13C NMR (100 MHz, CDCl3), d (ppm): 10.5 (C-23), 18.8 (C-27), 22.5 (C-26), 29.0 (C-15), 29.9 (C-12), 30.2 (C-30), 30.6 (C-21), 31.0 (C-17), 31.2 (C-19), 32.1 (C-28), 33.6 (C-11), 34.3 (C-22), 36.1 (piperazine 2 CH2), 36.5 (C-16), 38.5 (C-25), 39.9 (C-13), 40.3 (C-20), 43.1 (C-9), 45.0 (C-18), 45.1 (C-14), 53.6 (NCH2CH2N), 54.3 (NCH2CH2N), 55.7 (morpholine 2 CH2), 56.5 (piperazine 2 CH2), 67.0 (morpholine 2 CH2), 117.3 (C-4), 118.2 (C-7), 119.7 (C-1), 127.6 (C-5), 134.2 (C-6), 146.2 (C-3), 165.0 (C-10), 170.5 (C-8), 178.5 (C]O), 178.6 (C]O). HR-ESI-MS: m/z [MþH]þ calcd for C39H58N3O4: 632.4422; found: 632.4420; HPLC: tR 13.17 min, PHPLC 98.1%. 4.3.8. 3h: 3-Hydroxy-9b,13a-dimethyl-2-oxo-24,25,26trinoroleana-1(10),3,5,7-tetraen-29-oic piperazine, N-(2-hydroxyl) ethyl Red powder, yield, 50%; mp 150e154 C; 1H NMR (400 MHz, CDCl3), d (ppm): 7.03 (d, 1H, J ¼ 4.4 Hz, H-6), 6.53 (s, 1H, H-1), 6.36 (d, 1H, J ¼ 4.8 Hz, H-7), 3.65 (m, 6H, piperazine 2 CH2, CH2CH2OH), 2.56e2.48 (m, 6H, piperazine 2 CH2, CH2CH2OH), 2.22 (s, 3H, CH323), 1.46 (s, 3H, CH3-25), 1.29 (s, 3H, CH3-26), 1.28 (s, 3H, CH3-30), 1.14 (s, 3H, CH3-28), 0.60 (s, 3H, CH3-27); 13C NMR (100 MHz, CDCl3), d (ppm): 10.5 (C-23), 18.8 (C-27), 22.5 (C-26), 29.0 (C-15), 29.2 (C-12), 29.9 (C-30), 30.2 (C-21), 30.6 (C-17), 31.0 (C-19), 32.1 (C-28), 33.6 (C-11), 34.4 (C-22), 36.2 (piperazine 2 CH2), 36.5 (C16), 38.5 (C-25), 39.1 (C-13), 40.3 (C-20), 43.1 (C-9), 45.0 (C-18), 45.1 (C-14), 52.9 (piperazine 2 CH2), 58.0 (CH2CH2OH), 59.6 (CH2CH2OH), 117.4 (C-4), 118.6 (C-7), 119.6 (C-1), 127.5 (C-5), 134.4 (C-6), 146.3 (C-3), 164.9 (C-10), 170.3 (C-8), 176.2 (C]O), 178.5 (C] O). HR-ESI-MS: m/z [MþH]þ calcd for C35H51N2O4: 563.3843; found: 563.3833; HPLC: tR 10.48 min, PHPLC 99.5%. 4.3.9. 3i: 3-Hydroxy-9b,13a-dimethyl-2-oxo-24,25,26trinoroleana-1(10),3,5,7-tetraen-29-oic piperazine, N-(2dimethylamino)ethyl Red powder, yield, 37%; mp 127e132 C; 1H NMR (400 MHz, CDCl3), d (ppm): 7.02 (d, 1H, J ¼ 4.8 Hz, H-6), 6.52 (s, 1H, H-1), 6.35 (d, 1H, J ¼ 4.8 Hz, H-7), 2.48 (m, 4H, piperazine 2 CH2), 2.26 (m, 8H, piperazine 2 CH2, NCH2CH2N), 2.21 (s, 3H, CH3-23), 1.45 (s, 3H, CH3-25), 1.29 (s, 3H, CH3-26), 1.27 (s, 3H, CH3-30), 1.25 (s, 6H, 2 NCH3), 1.14 (s, 3H, CH3-28), 0.59 (s, 3H, CH3-27);13C NMR (CDCl3, 100 MHz), d (ppm): 10.5 (C-23), 18.8 (C-27), 22.5 (C-26), 29.1 (C-15), 29.9 (2 NCH3), 30.2 (C-12), 30.7 (C-30), 31.0 (C-21), 31.2 (C-17), 32.1 (C-19), 33.6 (C-28), 34.5 (C-11), 36.2 (22), 36.5 (C-16), 38.5 (C25), 39.9 (C-13), 40.3 (C-20), 43.2 (C-9), 45.0 (C-18), 45.1 (C-14), 45.9 (2 NCH2), 53.6 (2 NCH2), 56.5 (NCH2), 56.7 (NCH2), 117.4 (C-4), 118.5 (C-7), 119.6 (C-1), 127.6 (C-5), 134.4 (C-6), 146.2 (C-3), 164.8 (C10), 170.2 (C-8), 176.1 (C-28), 178.6 (C-2). HR-ESI-MS: m/z [MþH]þ calcd for C37H56N3O3: 590.4316; found: 590.4312; HPLC: tR 12.10 min, PHPLC 97.7%. 4.4. Crystallographic studies X-ray single-crystal diffraction data for compound 3a was collected on a Bruker SMART APEX CCD diffractometer at 294 K using CuKa radiation (l ¼ 1.54184 Å) by the u scan mode. The program SAINT was used for integration of the diffraction profiles. The structure was solved by direct methods using the SHELXS
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program of the SHELXTL package and refined by full-matrix leastsquares methods with SHELXL [28]. The corrections for LP factors were applied. All non-hydrogen atoms of compound 3a were refined with anisotropic thermal parameters. All hydrogen atoms were generated theoretically onto the parent atoms and refined isotropically with fixed thermal factors. 4.5. Anticancer assay The anti-proliferative activity of the title compounds against the four cell lines SGC-7901, SMMC-7721, MGC-803 and HepG-2 were evaluated using a standard MTT-based colorimetric assay. Ten thousand corresponding cells per well were seeded into 96-well plates (Corning, New York, USA) and incubated at 37 C, 5% CO2 for 24 h. And then 100 mL a series of concentration of drugcontaining medium were dispensed into wells to maintain the final concentration as 60, 20, 6.67, 2.22, 0.74, 0.25 and 0.082 mg/mL. Each concentration was in triplicate, and 5-fluorouracil (SigmaeAldrich, St. Louis, USA) and AMD were used as the positive control. After 48 h incubation, cell survival was determined by the addition of 25 mL MTT (SigmaeAldrich, St. Louis, USA) work solution (5 mg/mL MTT dissolved in Phosphate Buffer Solution (PBS)). After post-incubation at 37 C for 4 h, the medium was discarded following by adding 100 mL DMSO (SigmaeAldrich, St. Louis, USA). The plates were then eddied for 10 min for complete dissolution. The optical absorbance was measured at 570 nm. The data represented the mean of three independent experiments in triplicate and were expressed as mean ± SD. The IC50 value was defined as the concentration at which 50% of the cells could survive. 4.6. Apoptosis assay For cell apoptosis analysis by annexin V-FITC/PI apoptosis detection kit (BestBio, China), cells in the logarithmic phase of growth were harvested in cold PBS and collected by centrifugation for 5 min at 1000 g. Cells were re-suspended at a density of 1 106 cells/mL in 1 binding buffer, stained with FITC-labeled annexin V and PI for 20 min and immediately analyzed on a BD FACSVerse Flow Cytometer. 4.7. Telomerase activity assay Compounds 1, 2 and 3ae3i were tested in a search for small molecule inhibitors of telomerase activity by using the TRAP-PCRELISA assay. In detail, the MGC-803 cells were firstly maintained in DMEM medium (GIBCO, New York, USA) supplemented with 10% fetal bovineserum (GIBCO, NewYork, USA), streptomycin (0.1 mg/ mL) and penicillin (100 IU/mL) at 37 C in a humidified atmosphere containing 5% CO2. After trypsinization, 5 104 cultured cells in logarithmic growth were seeded into T25 flasks (Corning, New York, USA) and cultured to allow to adherence. The cells were then incubated with Staurosporine (Santa Cruz, Santa Cruz, USA) and the drugs with a series of concentration as 60, 20, 6.67, 2.22, 0.74, 0.25 and 0.0821 g/mL, respectively. After 24 h treatment, the cells were harvested by cell scraper orderly following by washed once with PBS. The cells were lysed in 150 mL RIPA cell lysis buffer (Santa Cruz, Santa Cruz, USA), and incubated on ice for 30 min. The cellular supernatants were obtained via centrifugation at 12,000 g for 20 min at 4 C and stored at 80 C. The TRAP-PCR-ELISA assay was performed using a telomerase detection kit (Roche, Basel, Switzerland) according to the manufacturer's protocol. In brief, 2 mL of cell extracts were mixed with 48 mL TRAP reaction mixtures. PCR was then initiated at 94 C, 120 s for predenaturation and performed using 35 cycles each consisting of 94 C for 30 s, 50 C for 30 s, 72 C for 90 s. Then 20 mL of PCR products were hybridized to a
digoxigenin (DIG)-labeled telomeric repeat specific detection probe. And the PCR products were immobilized via the biotinlabeled primer to a streptavidin-coated microtiter plate subsequently. The immobilized DNA fragment were detected with a peroxidase-conjugated anti-DIG antibody and visualized following addition of the stop regent. The microtitre plate was assessed on TECAN Infinite M200 microplate reader (Mannedorf, Switzerland) at a wavelength of 490 nm, and the final value were presented as mean ± SD. 4.8. General procedure for molecular docking In this study, a three-dimension human telomerase model and €dinger's IFD (Induced Fit Docking) were used to model in silico Schro the binding poses of our designed compounds with hTERT. Before doing the IFD, the structural errors of the model was first corrected manually and then the complex was treated by Protein Prepared €dinger. Wizard of Schro IFD is allowing incorporation of the protein and ligand flexibility in the docking protocol, which is consisted of the following steps: (i) constrained minimization of the protein with an RMSD cutoff of 0.18 Å; (ii) initial Glide docking of the ligand using a softened potential (Van der Waals radii scaling); (iii) one round of Prime sidechain prediction for each protein/ligand complex, on residues within defined distance of any ligand pose; (iv) prime minimization of the same set of residues and the ligand for each protein/ligand complex pose; (v) Glide re-docking of each protein/ligand complex structure within a specified energy of the lowest energy structure; (vi) estimation of the binding energy (IFDScore) for each output pose. All docking calculations were run in the extra precision (XP) mode of Glide. The center of the grid box of the hTERT was defined by two residues in the active site: LYS710 and LYS902. The size of the grid box was set to 15 Å. Default values were used for all other parameters. A further 10 ns MD simulation was submitted using a docking pose which can account for the limited SAR of BIBR1532 and BIBR1591 by employing the program of Desmond, which was developed at D. E. Shaw Research to perform high-speed molecular dynamics simulations of biological systems on conventional commodity clusters. All suggested values of Desmond were employed to run this 10 ns MD simulation. The MD simulation include the following steps: (i) a solvated system for simulation was generated; (ii) positive or negative counter ions were distributed to neutralize the system and additional ions (Naþ and Cl) to were introduced to set the desired ionic strength; (iii) OPLS-AA force field parameters were assigned to the entire molecular system; (iv) the system was relaxed or minimized; (v) the Desmond simulation was run. Compound 3c was docked into the active site of the modeled hTERT-BIBR1532 complex by employing the docking program of €dinger to evaluate its binding mode and binding Glide SP of Schro affinity. 4.9. The measurement of log P The octanol/water partition coefficient is defined as the ratio of a chemical's concentration in the octanol phase to its concentration in the aqueous phase of a two-phase octanol/water system. The logarithm of the ratio of the concentrations of the chemical in the solvents is called log P: the log P value is also known as a measure of lipophilicity. The shake-flask method is a classical and most reliable method of log P determination, which consists of dissolving some of the solute in question in a volume of octanol and water, then measuring the concentration of the solute in each solvent. HPLC method was used to measure the distribution of the solute, then the log P value was calculated by the following formula.
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log Poct=wat ¼ log
½soluteoc tan ol ½solutewater
Acknowledgments The authors wish to thank the National Natural Science Foundation of China (No. 21272008, 20802003), Science and Technological Fund of Anhui Province for Outstanding Youth (1408085J04), Anhui Provincial Natural Science Foundation (1308085MH137). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2015.03.039. References [1] T.M. Nakamura, G.B. Morin, K.B. Chapman, S.L. Weinrich, W.H. Andrews, J. Lingner, C.B. Harley, T.R. Cech, Science 277 (1997) 955e959. [2] P. Martínez, M.A. Blasco, Nat. Rev. Cancer 11 (2011) 161e176. [3] S. Sandin, D. Rhodes, Curr. Opin. Struct. Biol. 25 (2014) 104e110. [4] J.W. Shay, S. Bacchetti, Eur. J. Cancer 33 (1997) 787e791. [5] C. Autexier, N. Lue, Annu. Rev. Biochem. 75 (2006) 493e517. [6] X.Q. Wu, C. Huang, X. He, Y.Y. Tian, D.X. Zhou, Y. He, X.H. Liu, J. Li, Cell. Signal. 25 (2013) 2462e2468. [7] J. Ye, V.M. Renault, K. Jamet, E. Gilson, Nat. Rev. Genet. 15 (2014) 491e503. [8] W.C. Hahn, S.A. Stewart, M.W. Brooks, S.G. York, E. Eaton, A. Kurachi, R.L. Beijersbergen, J.H. Knoll, M. Meyerson, R.A. Weinberg, Nat. Med. 5 (1999) 1164e1170.
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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Design and synthesis of celastrol derivatives as anticancer agents Wen-Jian Tang a, 1, Jing Wang a, 1, Xu Tong a, Jing-Bo Shi a, Xin-Hua Liu a, b, *, Jun Li a, ** a b
School of Pharmacy, Anhui Medical University, Hefei, 230032, PR China State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 January 2015 Received in revised form 16 March 2015 Accepted 17 March 2015 Available online 18 March 2015
A series of celastrol derivatives as potential telomerase inhibitors were designed and synthesized. The bioassays demonstrated that title compounds displayed potent anticancer activities against SGC-7901, SMMC-7721, MGC-803 and HepG-2 cell lines, among them, compounds 3c and 3d which containing hydrophilicity moieties exhibited high anti-proliferative activities (IC50 ¼ 0.10e1.22 mM). The preliminary mechanism of antitumor action indicated that title compound 3c could induce significant SMMC-7721 cells apoptosis. A modified TRAP assay showed that compounds 3c and 3d displayed the most potent inhibitory activity with IC50 values at 0.11 and 0.34 mM, respectively. And there was a good correlation between telomerase inhibition and anti-proliferative inhibition of SMMC-7721 cells. Moreover, molecular docking indicated that the active compound 3c was nicely bound into the telomerase hTERT active site, hydrophobic, van der Waals and two hydrogen bond interactions with conserved residues ASP 628 and TYR 949 were found. © 2015 Elsevier Masson SAS. All rights reserved.
Keywords: Celastrol derivatives Anticancer activity Telomerase Cell apoptosis
1. Introduction Telomerase plays a pivotal role in bypassing cell senescence and maintaining telomere homeostasis. Telomerase reverse transcriptase (TERT) is a catalytic subunit of the enzyme telomerase [1e3]. TERT has been reported to be over-expressed in more than 90% of cancer cells, while most normal tissues and cells contain inactivated telomerase, thereby, telomerase plays a critical role in sustained proliferation and survival potentials of various cancer cells [4,5]. Numerous evidences have suggested that TERT could modulate the expression of numerous genes including cell cycle regulation and cellular signaling [6,7]. Therefore, targeting TERT is an important strategy for the development of cancer agents [8,9]. It was of our interest to utilize rational chemical approaches and discover novel natural compounds with anticancer as telomerase inhibitors from Traditional Chinese Medicine (TCM). Focused on telomerase TERT (pdb: 3DU6), we disclosed the X-ray crystal structure of some molecule inhibitors bound to active site of TERT [6,10,11], based on this, in our previous study, a compound 18aGAMG (pentacyclic triterpene scaffold, Fig. 1A) inhibited the
* Corresponding author. School of Pharmacy, Anhui Medical University, Hefei, Anhui 230032, PR China. ** Corresponding author. E-mail addresses: [email protected] (X.-H. Liu), [email protected] (J. Li). 1 W.J.T and J.W. contributed equally to this work. http://dx.doi.org/10.1016/j.ejmech.2015.03.039 0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.
expression of TERT and with selectivity activity against tumor cells versus human somatic cells was discovered [12]. But, unfortunately, the activity of this compound against to tumor cell lines is not high enough, herein, in continuation to extend our research on telomerase inhibitors, we began to look for other types of pentacyclic triterpene scaffolds according to above findings. Among these compounds, celastrol (Fig. 1B) is an active compound isolated from the root extracts of TCM Tripterygium wilfordii (Thunder of God Vine). Celastrol is a pentacyclic triterpenoid and belongs in the family of quinone methides. Celastrol has been investigated widely for its anti-inflammatory and anticancer activity [13,14]. This triterpene modulates multiple molecular targets such as Hsp90-Cdc37, TNF-a, NF-kB, VEGF, Akt, CXCR4, proinflammatory cytokines and chemokines involved playing a major role in all three steps (initiation, proliferation and progression) of carcinogenesis [15,16]. So far, celastrol has been shown to have beneficial effects on a variety of cancers in vitro and in vivo, including pancreatic cancer, hepatocellular carcinomas, squamous cancer, and prostate cancer [17e20], suggesting it might be developed as a potential cancer treatment. Although celastrol has important pharmacological activity, its poor water solubility and high toxicity restricts its application. Thus, some structure modifications were carried out to improve solubility or reduce toxicity, which focus on either the esterification or amidification of 20-carboxylic acid or the reduction products of A/B rings of celastrol [21e26]. Structural modifications at the C-2, 3
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167
O HO H
O
COOH HOOC HO HO
H O O OH
H
A: 18α-GAMG
H
Extract
COOH HO
O
H
HO
B: Celastrol
Title Pentacyclic triterpene scaffold
Fig. 1. The general design strategy in this study.
positions were reported as the inducers of heat shock response, while those at C-6 position displayed better anticancer activity. However, previous studies also suggested that the intact quinone methide moiety was crucial for its cytotoxic activity in cancer cell lines and neuroprotective effect [21,23]. In this study, based on improving celastrol water solubility and to further understand the preliminary structureeactivity relationship (SAR) against cancer cells, we rationally designed and synthesized a series of celastrol derivatives with the intact quinone methide group. The anti-proliferative activity and telomerase inhibitive activity of these derivatives were evaluated. 2. Results and discussion 2.1. Chemistry Carboxylic acid can be transferred to ether by alkylation reaction with the catalysis of alkali. Compound 2 was obtained by the esterification at 20-carboxylic acid of celastrol (1) in the presence of iodoethane and catalyst NaHCO3 in anhydrous DMF with 72% yield. Amides are commonly formed via reactions of an “activated” carboxylic acid with an amine. Compounds 3a~3i were obtained from amide condensation reaction catalyzed by EDC∙HCl, HOBt and TEA in anhydrous CH2Cl2 conditions with 26%~56% yield (Scheme 1). The products were purified by extraction, column chromatography and recrystallization, and their structures were confirmed by 1H NMR, 13C NMR and HRMS according to the literature [27]. Purify was evaluated under HPLC conditions and exceeded 96%. 2.2. Crystal structure analysis The structure of compound 3a was determined by X-ray crystallography. Crystal data of 3a: Clear light orange crystals obtained from EtOH/H2O, yield, 77%; mp 133e135 C; C30H41NO3, M ¼ 463.64, Monoclinic, space group P21; a ¼ 11.6031(5), b ¼ 8.1736(2), c ¼ 13.9701(7) (Å); a ¼ 90, b ¼ 106.252(5), g ¼ 90, V ¼ 1271.96(10) Å3, T ¼ 294 K, Z ¼ 2, Dc ¼ 1.211 g/cm3, F(000) ¼ 504, Reflections collected/Independent reflections ¼ 4621/3696, Data/ restraints/parameters ¼ 4621/1/315, Goodness of fit on F2 ¼ 1.045, Fine, R1 ¼ 0.0480, wR(F2) ¼ 0.1248. The molecular structure of compound 3a was shown in Fig. 2.
The absolute configuration of compound 3a was determined using CuKa radiation (l ¼ 1.54184 Å), that is C5(S), C11(S), C15(R), C17(S), C20(R) and C27(S). In compound 3a, rings A and B showed plane conformations, while the other rings showed mixed forms between envelope and half-chair conformations with atoms in rings C, D and E, respectively. Rings D and E were cis-form, which was similar to that of 18b-GAMG [12]. Crystallographic data (excluding structure factors) for the structure had been deposited with the Cambridge Crystallographic Data Center as supplementary publication No. CCDC 1034724 [28]. 2.3. In vitro anticancer activity All title compounds were evaluated for their anticancer activities in vitro against SGC-7901 (human gastric cancer cells), SMMC7721 (human hepatoma cells), MGC-803 (human gastric cancer cells) and HepG-2 (human hepatoma cells) cell lines, also included the activity of references 5-Fluorouracil and doxorubicin (AMD). The cells were allowed to proliferate in presence of tested material for 48 h, and the results are reported in terms of IC50 values (Table 1). Celastrol and its derivatives showed remarkable antiproliferative effects. Among them, compounds 1, 3c, 3d and 3i displayed potent inhibitory activities (IC50 1 mM, as 1, 3c, 3d, IC50 ¼ 0.15, 0.16, 0.10 mM for SGC-7901 cells; as 1, 3c, 3d, IC50 ¼ 0.58, 0.30, 0.61 mM for SMMC-7721 cells; as 3c, 3i, IC50 ¼ 0.39, 1.00 mM for MGC-803 cells; as 3c, 3d, IC50 ¼ 0.61, 0.28 mM for HepG-2 cells, respectively), which were more potent than the positive control 5Fluorouracil and AMD. Besides, compound 3h showed potent anticancer activities against SGC-7901 and SMMC-7721 cell lines, surpassing that of the positive control 5-Fluorouracil. Subsequently preliminary SAR studies were performed to determine how the substituent at 20-carboxylic acid affected the anticancer activity. Celastrol without any substituent group also showed good anti-proliferative activity. Firstly, the esterification of 20-carboxylic acid to form compound 2 showed lowest anticancer activities, whose IC50 values (9.28e15.19 mM) was lower than those of other compounds and the positive control. Secondly, the amidification of 20-carboxylic acid to form compounds 3a~3i displayed different anticancer activity (IC50 ¼ 0.10e19.97 mM). Compounds 3c, 3d with b-hydroxyl-ethylamide substituent exhibited potent anticancer activity against all four cell lines, while compounds 3h,
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O
O OH
R
A or B
H
O
H
O
HO
HO
2, 3
1
2: R = -OCH2CH3; 3a: R = -NHCH3; 3b: R = -N(CH3)2; 3c: R = -NHCH2CH2OH 3d: R = -NH-i-Propanol; 3e: R = -NH(CH2)3CH3; 3f: R =
N
O
; 3g: R =
N
N
N
O
; 3h: R =
N
N
OH
; 3i: R =
N
N
N
Reagents and conditions: (A) NaHCO3, DMF, iodoethane, rt; (B) EDC·HCl, HOBt, TEA, CH2Cl2. Scheme 1. Synthesis of title compounds 2 and 3.
Table 1 In vitro anticancer activity of the synthesized compounds against SGC-7901, SMMC7721, MGC-803 and HepG2 cell lines.a Compound
IC50 (mM)b SGC-7901
Fig. 2. ORTEP drawing of compound 3a.
3i showed moderate anticancer activity. Finally, compared to celastrol, compounds 3c, 3d increased 4.0, 1.3 and 6.6, 14.3 times anti-proliferative activity for MGC-803 and HepG-2 cells, respectively.
1 2 3a 3b 3c 3d 3e 3f 3g 3h 3i 5- Fluorouracilc AMDc
0.15 11.50 9.38 10.50 0.16 0.10 4.90 8.74 8.10 3.81 1.33 6.12 1.45
± ± ± ± ± ± ± ± ± ± ± ± ±
0.07 2.44 2.11 1.91 0.02 0.02 0.45 1.19 2.01 0.51 0.41 0.89 0.09
SMMC-7721 0.58 9.28 7.62 19.97 0.30 0.61 6.88 10.70 12.32 1.84 2.58 4.87 1.22
± ± ± ± ± ± ± ± ± ± ± ± ±
0.18 0.99 1.61 1.66 0.06 0.07 0.91 2.28 2.27 0.51 0.54 0.27 0.12
MGC-803 1.55 12.00 24.10 6.30 0.39 1.22 3.55 30.70 13.40 10.30 1.00 3.58 1.19
± ± ± ± ± ± ± ± ± ± ± ± ±
0.25 2.10 2.70 0.98 0.01 0.08 0.87 4.11 1.33 2.10 0.17 0.40 0.07
HepG2 4.01 15.19 10.60 11.20 0.61 0.28 3.07 22.70 16.38 8.70 5.21 5.86 0.87
± ± ± ± ± ± ± ± ± ± ± ± ±
1.21 1.21 2.45 0.98 0.11 0.07 0.51 4.87 2.15 2.12 0.44 0.73 0.10
Negative control DMSO, no activity. a The data represented the mean of three experiments in triplicate and were expressed as means ± SD; only descriptive statistics were done in the text. b The IC50 value was defined as the concentration at which 50% survival of cells was observed. The results are listed in the table. c Used as a positive control.
2.5. Telomerase activity assay 2.4. Induction of apoptosis by compound 3c The annexin V-FITC/PI apoptosis detection kit was used to determine whether compound 3c meditated inhibition of growth and proliferation was associated with apoptosis. As shown in Fig. 3, only a small percentage of untreated SMMC-7721 (2.0%) cells bound with annexin V-FITC. In contrast, the percentage of annexin V-FITC binding SMMC-7721 cells significantly increased after treatment with 0.5 mM compound 3c (2.0%e61.2%, p < 0.05). In a word, data points were dispersed and shifted to the Q3 side when SMMC-7721 cells treated with compound 3c, indicating that the cells moved to the early apoptotic stage. The experimental results demonstrated that compound 3c induced apoptosis of SMMC-7721 cells.
The synthesized compounds were assayed for telomerase inhibition using an MGC-803 cell extract, and BIBR1532 was selected as the reference compound. The results were presented as mean ± SD. As shown in Table 2, the tested compounds displayed good telomerase inhibitory. Among them, compounds 3c and 3d displayed strong telomerase inhibitory activity with IC50 values of 0.11 and 0.34 mM respectively, which was superior to that of the positive control BIBR1532. Then, an analysis between telomerase inhibition and inhibition of SMMC-7721 cellular proliferation indicated that there was a good correlation with an R value of 0.968, as evidenced in Fig. 4. This demonstrated that the potent inhibitory effects of title compounds on the cell proliferation assay were causally related to
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169
Fig. 3. Compound 3c induced apoptosis in SMMC-7721 using annexin V-FITC/PI. (A)e(B) Treatment with 0 and 0.5 mM compound 3c for 48 h, respectively. The experiments were repeated three times and representative photographs are shown. A: normal SMMC-7721, B: SMMC-7721þ0.5 mM compound 3c. Q1: necrotic cells, Q2: viable apoptotic cells, Q3: non-viable apoptotic cells, Q4: normal cells.
further optimize the structure of celastrol with anticancer activity as potential telomerase inhibitors.
Table 2 LogP and inhibitory effects of title compounds against telomerase. Compound
logP
IC50 (mM) telomerasea
1 2 3a 3b 3c 3d 3f 3g 3h 3i BIBR1532b
4.38 4.98 3.96 4.20 3.44 3.49 3.80 3.50 3.44 3.95
0.78 18.55 15.10 21.01 0.11 0.34 8.01 15.17 2.01 1.41 0.41
a b
± ± ± ± ± ± ± ± ± ± ±
0.17 1.42 1.61 1.95 0.02 0.07 0.81 1.80 0.33 0.20 0.05
Telomerase supercoiling activity. BIBR1532 is reported as a control.
2.6. The octanol/water partition coefficient (log P) In order to know whether is the polarity of celastrol derivative affects its anti-proliferative activity in this study. Theoretical octanol/water partition coefficient (log P) of synthesized compounds was measured by chemical software ChemDraw 12.0. As showed in Table 2, the inhibitory activity of synthesized compounds was associated with log P value. Compared to celastrol (1, log P ¼ 4.38), compounds 3c and 3d with b-hydroxyl-ethylamide moiety showed better hydrophilic (log P ¼ 3.44 and 3.59, respectively), which displayed higher anticancer activity. 2.7. Molecular docking
their telomerase inhibitory activities. These results suggested that compounds 3c and 3d were excellent anticancer agents with potent telomerase inhibitory, which also pointed out the direction for us to
A three-dimension human telomerase model [29] and an advanced docking method e IFD (Induced Fit Docking) of €dinger were employed to explore the binding mode of Schro BIBR1532, a potent hTERT inhibitor. The docked complex hTERTeBIBR1532 was then done a 10 ns MD simulation. Compound 3c was docked into the active site of the modeled hTERTeBIBR1532 complex to see its putative binding pose. As shown in Fig. 5, a typical docking pose demonstrates that the flexible side chain of compound 3c forms two hydrogen bonds with the receptor: one is between the hydroxyl group and residue of ASP 628; another is between the hydroxyl group and residue of TYR 949. The hydrophobic and rigid fused ring of compound 3c, whereas, is inserted into the active site of hTERT and has hydrophobic and van der Waals interactions with the receptor. 3. Conclusion
Fig. 4. The linear regression results of cell lines versus kinases to explore the correlation between pIC50's for the inhibition of telomerase and SMMC-7721 cellular proliferation, R ¼ 0.968.
In conclusion, based on our previous study, focused on pentacyclic triterpene moiety, a series of celastrol derivatives as potential telomerase inhibitors were designed and synthesized. In vitro bioassays demonstrated that celastrol derivatives displayed potent anticancer activities against SGC-7901, SMMC-7721, MGC-803 and HepG-2 cell lines, among them, compounds 3c and 3d exhibited high anti-proliferative activities (IC50 ¼ 0.10e1.22 mM), which hydrophilicity might be one of the reasons for high anticancer activity. The telomerase assay showed that there was a good correlation
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CDCl3), d (ppm): 6.99 (d, 1H, J ¼ 4.7 Hz, H-6), 6.50 (s, 1H, H-1), 6.32 (d, 1H, J ¼ 4.8 Hz, H-7), 3.94 (m, 2H, OCH2CH3), 2.46 (d, 1H, J ¼ 10.5 Hz, H-19a), 2.18 (s, 3H, CH3-23), 1.42 (s, 3H, CH3-25), 1.23 (s, 3H, CH3-26), 1.17 (t, 3H, J ¼ 4.8 Hz, OCH2CH3), 1.14 (s, 3H, CH3-30), 1.07 (s, 3H, CH3-28), 0.53 (s, 3H, CH3-27); 13C NMR (100 MHz, CDCl3), d (ppm): 10.5 (C-23), 14.2 (OCH2CH3), 18.3 (C-27), 21.8 (C26), 28.8 (C-15), 29.8 (C-12), 30.0 (C-30), 30.8 (C-17), 30.9 (C-21), 31.8 (C-19), 33.0 (C-28), 33.7 (C-11), 34.9 (C-22), 36.5 (C-16), 38.4 (C-25), 39.6 (C-13), 40.4 (C-20), 43.1 (C-9), 44.4 (C-18), 45.2 (C-14), 60.5 (OCH2CH3), 117.4 (C-4), 118.3 (C-7), 119.7 (C-1), 127.6 (C-5), 134.4 (C-6), 146.2 (C-3), 164.9 (C-10), 170.4 (C-8), 178.3, 178.6 (2 C]O). HR-ESI-MS: m/z [MþH]þ calcd for C31H43O4: 479.3156; found: 479.3157; HPLC: tR 4.40 min, PHPLC 99.7%. 4.3. General procedure for the synthesis of title compound 3 € dinger. Fig. 5. Binding mode of compound 3c to hTERT yielded by Glide 5.9 of Schro
between telomerase inhibition and anti-proliferative inhibition of SMMC-7721 cells, and compounds 3c and 3d displayed the most potent inhibitory activity with IC50 values at 0.11 and 0.34 mM respectively. Molecular docking indicated that the active compound 3c was nicely bound to telomerase hTERT via two hydrogen bonds and van der Waals interactions. Moreover, the mechanism of antitumor action indicated that title compound 3c could induce significant cell apoptosis in SMMC-7721 cells. These results support further studies to optimize structure of celastrol as telomerase inhibitors in the future. 4. Experimental section 4.1. Chemistry All reagents were purchased from commercial sources and were used without further purification. Melting points (uncorrected) were determined on a XT4MP apparatus (Taike Corp., Beijing, China). 1H NMR and 13C NMR spectra were recorded on Bruker AV400 or AV-300 MHz instruments in CDCl3 using tetramethylsilane (TMS) as the internal standard. Chemical shifts are reported in ppm (d). Coupling constants are reported in hertz. The multiplicity is defined by s (singlet), d (doublet), t (triplet), or m (multiplet). Mass spectra were recorded on a LTQ-Orbitrap XL (Thermo-Fisher Scientific, LLC). Column and thin-layer chromatography (CC and TLC, resp.) were performed on silica gel (200e300 mesh) and silica gel GF254 (Qingdao Marine Chemical Factory) respectively. HPLC was performed on a Shimadzu LC-20AT system equipped with an autoinjector and a UV detector set at 254 nm. Shimadzu software was used to calculate peak areas. Compounds were separated on a Spherisorb C18 column from DEAIC (Dalian, China). The mobile phase was a mixture of MeOH/H2O (95:5 / 85:15) for compounds 3a-3i. HPLC retention times (HPLC tR) were obtained at a flow rate of 1.0 mL/min. 4.2. The synthesis of title compound 2 To a DMF (2 mL) solution of celastrol (45 mg, 0.10 mmol) was added NaHCO3 (19 mg, 0.22 mmol) and iodoethane (18 mL, 0.22 mmol). The reaction mixture was stirred for 3 h at room temperature, then washed twice with water, dried with Na2SO4, filtered, and concentrated in vacuo. The residue was purified by crystallization from ethanol to give title compound 2. 4.2.1. 3-Hydroxy-9b,13a-dimethyl-2-oxo-24,25,26-trinoroleana1(10),3,5,7-tetraen- 29-oic acid ethyl ester Red powder, yield, 72%; mp 116e119 C; 1H NMR (400 MHz,
To a CH2Cl2 (5 mL) solution of celastrol (45 mg, 0.10 mmol) was added EDC∙HCl (43 mg, 0.22 mmol), HOBt (30 mg, 0.22 mmol), and corresponding amine (2e3 eq.), the mixture was stirred 30 min at 0 C. After TEA (50 mL) was added, the reaction solution was stirred overnight at room temperature. The mixture was washed twice with water, dried with Na2SO4, filtered, and concentrated in vacuo. The residue was purified by chromatography on a silica gel column (dichloromethane/methanol, 10:0 / 10:1) to give title compounds 3ae3i. 4.3.1. 3a: 3-Hydroxy-9b,13a-dimethyl-2-oxo-24,25,26trinoroleana-1(10),3,5,7-tetraen-29-oic amide, N-methyl Red powder, yield, 49%; mp 131e134 C; 1H NMR (400 MHz, CDCl3), d (ppm): 6.98 (d, 1H, J ¼ 4.4 Hz, H-6), 6.50 (s, 1H, H-1), 6.31 (d, 1H, J ¼ 4.8 Hz, H-7), 5.75 (m, 1H, J ¼ 2.8 Hz, NH), 2.64 (d, 3H, J ¼ 2.8 Hz, NCH3), 2.43 (d, 1H, J ¼ 10.4 Hz, H-19a), 2.18 (s, 3H, CH323), 1.41 (s, 3H, CH3-25), 1.23 (s, 3H, CH3-26), 1.12 (s, 3H, CH3-30), 1.09 (s, 3H, CH3-28), 0.59 (s, 3H, CH3-27); 13C NMR (100 MHz, CDCl3), d (ppm): 10.5 (C-23), 18.3 (C-27), 21.9 (C-26), 26.7 (NCH3), 28.8 (C-15), 29.6 (C-12), 29.9 (C-30), 30.3 (C-21), 31.1 (C-17), 31.5 (C19), 31.8 (C-28), 33.7 (C-11), 33.8 (C-28), 36.6 (C-16), 38.4 (C-25), 39.5 (C-13), 40.5 (C-20), 43.2 (C-9), 44.6 (C-18), 45.3 (C-14), 117.3 (C4), 118.2 (C-7), 119.7 (C-1), 127.6 (C-5), 134.2 (C-6), 146.2 (C-3), 165.0 (C-10), 170.5 (C-8), 178.5 (C]O), 178.6 (C]O). HR-ESI-MS: m/z [MþH]þ calcd for C30H42NO3: 464.3159; found: 464.3153; HPLC: tR 8.80 min, PHPLC 99.7%. 4.3.2. 3b: 3-Hydroxy-9b,13a-dimethyl-2-oxo-24,25,26trinoroleana-1(10),3,5,7-tetraen-29-oic amide, N,N-dimethyl Red powder, yield, 33%; mp 141e144 C; 1H NMR (400 MHz, CDCl3), d (ppm): 7.04 (d, 1H, J ¼ 6.8 Hz, H-6), 6.54 (s, 1H, H-1), 6.36 (d, 1H, J ¼ 6.8 Hz, H-7), 3.20 (s, 3H, NCH3), 2.81 (s, 3H, NCH3), 2.42 (d, 1H, J ¼ 16.0 Hz, H-19a), 2.35 (d, 1H, J ¼ 14.1 Hz, H-19b), 2.22 (s, 3H, CH3-23), 1.46 (s, 3H, CH3-25), 1.28 (s, 6H, CH3-26, CH3-30), 1.14 (s, 3H, CH3-28), 0.54 (s, 3H, CH3-27); 13C NMR (100 MHz, CDCl3), d (ppm): 10.3 (C-23), 18.5 (C-27), 22.1 (C-26), 28.8 (C-15), 29.7 (C12), 30.0 (C-30), 30.4 (C-21), 30.8 (C-17), 31.0 (C-19), 32.0 (C-28), 33.5 (C-11), 34.4 (C-22), 36.0 (2 NCH3), 36.4 (C-16), 38.3 (C-25), 39.6 (C-13), 40.2 (C-20), 42.9 (C-9), 44.9 (C-18), 45.1 (C-14), 117.3 (C4), 118.3 (C-7), 119.4 (C-1), 127.4 (C-5), 134.4 (C-6), 146.0 (C-3), 164.8 (C-10), 170.2 (C-8), 176.9 (C]O), 178.3 (C]O). HR-ESI-MS: m/z [MþH]þ calcd for C31H44NO3: 478.3316; found: 478.3315; HPLC: tR 11.34 min, PHPLC 99.7%. 4.3.3. 3c: 3-Hydroxy-9b,13a-dimethyl-2-oxo-24,25,26trinoroleana-1(10),3,5,7-tetraen-29-oic amide, N-(2-hydroxy)ethyl Red powder, yield, 38%; mp 182e185 C; 1H NMR (400 MHz, CDCl3), d (ppm): 6.98 (d, 1H, J ¼ 4.6 Hz, H-6), 6.49 (s, 1H, H-1), 6.30 (d, 1H, J ¼ 4.6 Hz, H-7), 6.29 (s, 1H, NH), 3.61 (m, 2H, CH2OH), 2.41
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(d, 1H, J ¼ 10.4 Hz, H-19a), 2.18 (s, 3H, CH3-23), 1.40 (s, 3H, CH3-25), 1.22 (s, 3H, CH3-26), 1.14 (s, 3H, CH3-30), 1.09 (s, 3H, CH3-28), 0.61 (s, 3H, CH3-27); 13C NMR (100 MHz, CDCl3), d (ppm): 10.5 (C-23), 18.5 (C-27), 21.9 (C-26), 28.8 (C-15), 29.6 (C-12), 29.9 (C-30), 30.3 (C-21), 31.0 (C-17), 31.3 (C-19), 31.8 (C-28), 33.7 (C-11), 34.1 (C-22), 36.5 (C16), 38.4 (C-25), 39.5 (C-13), 40.5 (C-20), 42.7 (NHCH2), 43.2 (C-9), 44.5 (C-18), 45.2 (C-14), 62.2(CH2OH), 117.3 (C-4), 118.2 (C-7), 119.7 (C-1), 127.6 (C-5), 134.2 (C-6), 146.2 (C-3), 165.0 (C-10), 170.5 (C-8), 178.5 (C]O), 178.6 (C]O). HR-ESI-MS: m/z [MþH]þ calcd for C31H44NO4: 494.3265; found: 494.3266; HPLC: tR 6.60 min, PHPLC 99.4%. 4.3.4. 3d: 3-Hydroxy-9b,13a-dimethyl-2-oxo-24,25,26trinoroleana-1(10),3,5,7-tetraen-29-oic amide, N-(2-hydroxy) propyl Red powder, yield, 33%; mp 90e92 C; 1H NMR (300 MHz, CDCl3), d (ppm): 7.02 (d, 1H, J ¼ 6.9 Hz, H-6), 6.98 (s, 1H, OH), 6.53 (s, 1H, H-1), 6.34 (d, 1H, J ¼ 7.2 Hz, H-7), 5.85 (d, 1H, J ¼ 6.9 Hz, NH), 3.50 (m, 2H, CH2OH), 2.40 (d, 1H, J ¼ 15.8 Hz, H-19a), 2.21 (s, 3H, CH3-23), 1.45 (s, 3H, CH3-25), 1.26 (s, 3H, CH3-26), 1.25 (d, 3H, J ¼ 3.0 Hz, CHCH3), 1.16 (s, 3H, CH3-30), 1.11 (s, 3H, CH3-28), 0.67 (s, 3H, CH3-27). 13C NMR (75 MHz, CDCl3), d (ppm): 10.5 (C-23), 18.3 (CHCH3), 18.7 (C-27), 22.0 (C-26), 28.9 (C-15), 29.8 (C-12), 29.9 (C30), 30.4 (C-21), 31.0 (C-17), 31.8 (C-19), 32.1 (C-28), 33.7 (C-11), 34.2 (C-22), 36.5 (C-16), 38.4 (C-25), 39.6 (C-13), 40.5 (C-20), 43.2 (C-9), 44.6 (C-18), 45.2 (C-14), 47.9 (NHCH), 67.3 (CH2OH), 117.3 (C4), 118.3 (C-7), 119.8 (C-1), 127.6 (C-5), 134.3 (C-6), 146.2 (C-3), 164.9 (C-10), 170.2 (C-8), 178.6 (C]O), 178.7 (C]O). HR-ESI-MS: m/z [MþH]þ calcd for C32H46NO4: 508.3421; found: 508.3416; HPLC: tR 10.28 min, PHPLC 99.5%. 4.3.5. 3e: 3-Hydroxy-9b,13a-dimethyl-2-oxo-24,25,26trinoroleana-1(10),3,5,7-tetraen-29-oic amide, N-n-butyl Red powder, yield, 56%; mp 80e82 C; 1H NMR (300 MHz, CDCl3), d (ppm): 7.02 (d, 1H, J ¼ 6.3 Hz, H-6), 7.01 (s, 1H, OH), 6.53 (s, 1H, H-1), 6.35 (d, 1H, J ¼ 7.2 Hz, H-7), 5.73 (m, 1H, NH), 3.10 (m, 2H, CH2NH), 2.46 (d, 1H, J ¼ 14.3 Hz, H-19a), 2.21 (s, 3H, CH3-23), 1.44 (s, 3H, CH3-25), 1.26 (s, 3H, CH3-26), 1.15 (s, 3H, CH3-30), 1.12 (s, 3H, CH3-28), 0.86 (t, 3H, J ¼ 7.2 Hz, CH2CH3), 0.64 (s, 3H, CH3-27); 13C NMR (75 MHz, CDCl3), d (ppm): 10.4 (C-23), 14.0 (CH2CH3), 18.4 (C27), 20.4 (CH2CH3), 22.0 (C-26), 28.8 (C-15), 29.6 (C-12), 30.4 (C-30), 31.0 (C-21), 31.3 (C-17), 31.5 (CH2CH2CH3), 31.8 (C-19), 33.7 (C-28), 34.0 (C-11), 35.2 (C-22), 36.5 (C-16), 38.3 (C-25), 39.5, 39.6 (C-13, NHCH2), 40.4 (C-20), 43.2 (C-9), 44.5 (C-18), 45.2 (C-14), 117.2 (C-4), 118.2 (C-7), 119.7 (C-1), 127.6 (C-5), 134.3 (C-6), 146.2 (C-3), 165.0 (C10), 170.5 (C-8), 177.8 (C]O), 178.5 (C]O). HR-ESI-MS: m/z [MþH]þ calcd for C33H48NO3: 506.3629; found: 506.3613; HPLC: tR 15.80 min, PHPLC 99.7%. 4.3.6. 3f: 3-Hydroxy-9b,13a-dimethyl-2-oxo-24,25,26trinoroleana-1(10),3,5,7-tetraen-29-oic morpholine Red powder, yield, 37%; mp 133e137 C; 1H NMR (400 MHz, CDCl3), d (ppm): 6.99 (d, 1H, J ¼ 4.7 Hz, H-6), 6.50 (s, 1H, H-1), 6.32 (d, 1H, J ¼ 4.8 Hz, H-7), 3.61 (m, 8H, 2 NCH2, 2 OCH2), 2.28 (d, 1H, J ¼ 9.8 Hz, H-19a), 2.18 (s, 3H, CH3-23), 1.43 (s, 3H, CH3-25), 1.26 (s, 3H, CH3-26), 1.25 (s, 3H, CH3-30), 1.11 (s, 3H, CH3-28), 0.58 (s, 3H, CH3-27); 13C NMR (100 MHz, CDCl3), d (ppm): 10.5 (C-23), 18.9 (C27), 22.6 (C-26), 29.1 (C-15), 29.9 (C-12), 30.2 (C-30), 30.8 (C-21), 30.9 (C-17), 31.1 (C-19), 32.1 (C-28), 33.5 (C-11), 34.3 (C-22), 36.2 (2 NCH2), 36.4 (C-16), 38.5 (C-25), 39.8 (C-13), 40.3 (C-20), 43.1 (C-9), 44.9 (C-18), 45.0 (C-14), 66.6 (2 OCH2), 117.3 (C-4), 118.5 (C7), 119.7 (C-1), 127.6 (C-5), 134.3 (C-6), 146.2 (C-3), 164.7 (C-10), 170.1 (C-8), 176.4 (C]O), 178.5 (C]O). HR-ESI-MS: m/z [MþH]þ calcd for C33H46NO4: 520.3421; found: 520.3419; HPLC: tR 8.55 min, PHPLC 97.7%.
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4.3.7. 3g: 3-Hydroxy-9b,13a-dimethyl-2-oxo-24,25,26trinoroleana-1(10),3,5,7-tetraen-29-oic piperazine, N-(2morpholinyl)ethyl Red powder, yield, 26%; mp 118e122 C; 1H NMR (400 MHz, CDCl3), d (ppm): 6.99 (d, 1H, J ¼ 4.7 Hz, H-6), 6.50 (s, 1H, H-1), 6.32 (d, 1H, J ¼ 4.8 Hz, H-7), 3.68 (m, 8H, morpholine 4 CH2), 2.48e2.45 (m, 12H, piperazine 4 CH2, NCH2CH2N), 2.18 (s, 3H, CH3-23), 1.42 (s, 3H, CH3-25), 1.25 (s, 3H, CH3-26), 1.23 (s, 3H, CH3-30), 1.10 (s, 3H, CH3-28), 0.56 (s, 3H, CH3-27); 13C NMR (100 MHz, CDCl3), d (ppm): 10.5 (C-23), 18.8 (C-27), 22.5 (C-26), 29.0 (C-15), 29.9 (C-12), 30.2 (C-30), 30.6 (C-21), 31.0 (C-17), 31.2 (C-19), 32.1 (C-28), 33.6 (C-11), 34.3 (C-22), 36.1 (piperazine 2 CH2), 36.5 (C-16), 38.5 (C-25), 39.9 (C-13), 40.3 (C-20), 43.1 (C-9), 45.0 (C-18), 45.1 (C-14), 53.6 (NCH2CH2N), 54.3 (NCH2CH2N), 55.7 (morpholine 2 CH2), 56.5 (piperazine 2 CH2), 67.0 (morpholine 2 CH2), 117.3 (C-4), 118.2 (C-7), 119.7 (C-1), 127.6 (C-5), 134.2 (C-6), 146.2 (C-3), 165.0 (C-10), 170.5 (C-8), 178.5 (C]O), 178.6 (C]O). HR-ESI-MS: m/z [MþH]þ calcd for C39H58N3O4: 632.4422; found: 632.4420; HPLC: tR 13.17 min, PHPLC 98.1%. 4.3.8. 3h: 3-Hydroxy-9b,13a-dimethyl-2-oxo-24,25,26trinoroleana-1(10),3,5,7-tetraen-29-oic piperazine, N-(2-hydroxyl) ethyl Red powder, yield, 50%; mp 150e154 C; 1H NMR (400 MHz, CDCl3), d (ppm): 7.03 (d, 1H, J ¼ 4.4 Hz, H-6), 6.53 (s, 1H, H-1), 6.36 (d, 1H, J ¼ 4.8 Hz, H-7), 3.65 (m, 6H, piperazine 2 CH2, CH2CH2OH), 2.56e2.48 (m, 6H, piperazine 2 CH2, CH2CH2OH), 2.22 (s, 3H, CH323), 1.46 (s, 3H, CH3-25), 1.29 (s, 3H, CH3-26), 1.28 (s, 3H, CH3-30), 1.14 (s, 3H, CH3-28), 0.60 (s, 3H, CH3-27); 13C NMR (100 MHz, CDCl3), d (ppm): 10.5 (C-23), 18.8 (C-27), 22.5 (C-26), 29.0 (C-15), 29.2 (C-12), 29.9 (C-30), 30.2 (C-21), 30.6 (C-17), 31.0 (C-19), 32.1 (C-28), 33.6 (C-11), 34.4 (C-22), 36.2 (piperazine 2 CH2), 36.5 (C16), 38.5 (C-25), 39.1 (C-13), 40.3 (C-20), 43.1 (C-9), 45.0 (C-18), 45.1 (C-14), 52.9 (piperazine 2 CH2), 58.0 (CH2CH2OH), 59.6 (CH2CH2OH), 117.4 (C-4), 118.6 (C-7), 119.6 (C-1), 127.5 (C-5), 134.4 (C-6), 146.3 (C-3), 164.9 (C-10), 170.3 (C-8), 176.2 (C]O), 178.5 (C] O). HR-ESI-MS: m/z [MþH]þ calcd for C35H51N2O4: 563.3843; found: 563.3833; HPLC: tR 10.48 min, PHPLC 99.5%. 4.3.9. 3i: 3-Hydroxy-9b,13a-dimethyl-2-oxo-24,25,26trinoroleana-1(10),3,5,7-tetraen-29-oic piperazine, N-(2dimethylamino)ethyl Red powder, yield, 37%; mp 127e132 C; 1H NMR (400 MHz, CDCl3), d (ppm): 7.02 (d, 1H, J ¼ 4.8 Hz, H-6), 6.52 (s, 1H, H-1), 6.35 (d, 1H, J ¼ 4.8 Hz, H-7), 2.48 (m, 4H, piperazine 2 CH2), 2.26 (m, 8H, piperazine 2 CH2, NCH2CH2N), 2.21 (s, 3H, CH3-23), 1.45 (s, 3H, CH3-25), 1.29 (s, 3H, CH3-26), 1.27 (s, 3H, CH3-30), 1.25 (s, 6H, 2 NCH3), 1.14 (s, 3H, CH3-28), 0.59 (s, 3H, CH3-27);13C NMR (CDCl3, 100 MHz), d (ppm): 10.5 (C-23), 18.8 (C-27), 22.5 (C-26), 29.1 (C-15), 29.9 (2 NCH3), 30.2 (C-12), 30.7 (C-30), 31.0 (C-21), 31.2 (C-17), 32.1 (C-19), 33.6 (C-28), 34.5 (C-11), 36.2 (22), 36.5 (C-16), 38.5 (C25), 39.9 (C-13), 40.3 (C-20), 43.2 (C-9), 45.0 (C-18), 45.1 (C-14), 45.9 (2 NCH2), 53.6 (2 NCH2), 56.5 (NCH2), 56.7 (NCH2), 117.4 (C-4), 118.5 (C-7), 119.6 (C-1), 127.6 (C-5), 134.4 (C-6), 146.2 (C-3), 164.8 (C10), 170.2 (C-8), 176.1 (C-28), 178.6 (C-2). HR-ESI-MS: m/z [MþH]þ calcd for C37H56N3O3: 590.4316; found: 590.4312; HPLC: tR 12.10 min, PHPLC 97.7%. 4.4. Crystallographic studies X-ray single-crystal diffraction data for compound 3a was collected on a Bruker SMART APEX CCD diffractometer at 294 K using CuKa radiation (l ¼ 1.54184 Å) by the u scan mode. The program SAINT was used for integration of the diffraction profiles. The structure was solved by direct methods using the SHELXS
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program of the SHELXTL package and refined by full-matrix leastsquares methods with SHELXL [28]. The corrections for LP factors were applied. All non-hydrogen atoms of compound 3a were refined with anisotropic thermal parameters. All hydrogen atoms were generated theoretically onto the parent atoms and refined isotropically with fixed thermal factors. 4.5. Anticancer assay The anti-proliferative activity of the title compounds against the four cell lines SGC-7901, SMMC-7721, MGC-803 and HepG-2 were evaluated using a standard MTT-based colorimetric assay. Ten thousand corresponding cells per well were seeded into 96-well plates (Corning, New York, USA) and incubated at 37 C, 5% CO2 for 24 h. And then 100 mL a series of concentration of drugcontaining medium were dispensed into wells to maintain the final concentration as 60, 20, 6.67, 2.22, 0.74, 0.25 and 0.082 mg/mL. Each concentration was in triplicate, and 5-fluorouracil (SigmaeAldrich, St. Louis, USA) and AMD were used as the positive control. After 48 h incubation, cell survival was determined by the addition of 25 mL MTT (SigmaeAldrich, St. Louis, USA) work solution (5 mg/mL MTT dissolved in Phosphate Buffer Solution (PBS)). After post-incubation at 37 C for 4 h, the medium was discarded following by adding 100 mL DMSO (SigmaeAldrich, St. Louis, USA). The plates were then eddied for 10 min for complete dissolution. The optical absorbance was measured at 570 nm. The data represented the mean of three independent experiments in triplicate and were expressed as mean ± SD. The IC50 value was defined as the concentration at which 50% of the cells could survive. 4.6. Apoptosis assay For cell apoptosis analysis by annexin V-FITC/PI apoptosis detection kit (BestBio, China), cells in the logarithmic phase of growth were harvested in cold PBS and collected by centrifugation for 5 min at 1000 g. Cells were re-suspended at a density of 1 106 cells/mL in 1 binding buffer, stained with FITC-labeled annexin V and PI for 20 min and immediately analyzed on a BD FACSVerse Flow Cytometer. 4.7. Telomerase activity assay Compounds 1, 2 and 3ae3i were tested in a search for small molecule inhibitors of telomerase activity by using the TRAP-PCRELISA assay. In detail, the MGC-803 cells were firstly maintained in DMEM medium (GIBCO, New York, USA) supplemented with 10% fetal bovineserum (GIBCO, NewYork, USA), streptomycin (0.1 mg/ mL) and penicillin (100 IU/mL) at 37 C in a humidified atmosphere containing 5% CO2. After trypsinization, 5 104 cultured cells in logarithmic growth were seeded into T25 flasks (Corning, New York, USA) and cultured to allow to adherence. The cells were then incubated with Staurosporine (Santa Cruz, Santa Cruz, USA) and the drugs with a series of concentration as 60, 20, 6.67, 2.22, 0.74, 0.25 and 0.0821 g/mL, respectively. After 24 h treatment, the cells were harvested by cell scraper orderly following by washed once with PBS. The cells were lysed in 150 mL RIPA cell lysis buffer (Santa Cruz, Santa Cruz, USA), and incubated on ice for 30 min. The cellular supernatants were obtained via centrifugation at 12,000 g for 20 min at 4 C and stored at 80 C. The TRAP-PCR-ELISA assay was performed using a telomerase detection kit (Roche, Basel, Switzerland) according to the manufacturer's protocol. In brief, 2 mL of cell extracts were mixed with 48 mL TRAP reaction mixtures. PCR was then initiated at 94 C, 120 s for predenaturation and performed using 35 cycles each consisting of 94 C for 30 s, 50 C for 30 s, 72 C for 90 s. Then 20 mL of PCR products were hybridized to a
digoxigenin (DIG)-labeled telomeric repeat specific detection probe. And the PCR products were immobilized via the biotinlabeled primer to a streptavidin-coated microtiter plate subsequently. The immobilized DNA fragment were detected with a peroxidase-conjugated anti-DIG antibody and visualized following addition of the stop regent. The microtitre plate was assessed on TECAN Infinite M200 microplate reader (Mannedorf, Switzerland) at a wavelength of 490 nm, and the final value were presented as mean ± SD. 4.8. General procedure for molecular docking In this study, a three-dimension human telomerase model and €dinger's IFD (Induced Fit Docking) were used to model in silico Schro the binding poses of our designed compounds with hTERT. Before doing the IFD, the structural errors of the model was first corrected manually and then the complex was treated by Protein Prepared €dinger. Wizard of Schro IFD is allowing incorporation of the protein and ligand flexibility in the docking protocol, which is consisted of the following steps: (i) constrained minimization of the protein with an RMSD cutoff of 0.18 Å; (ii) initial Glide docking of the ligand using a softened potential (Van der Waals radii scaling); (iii) one round of Prime sidechain prediction for each protein/ligand complex, on residues within defined distance of any ligand pose; (iv) prime minimization of the same set of residues and the ligand for each protein/ligand complex pose; (v) Glide re-docking of each protein/ligand complex structure within a specified energy of the lowest energy structure; (vi) estimation of the binding energy (IFDScore) for each output pose. All docking calculations were run in the extra precision (XP) mode of Glide. The center of the grid box of the hTERT was defined by two residues in the active site: LYS710 and LYS902. The size of the grid box was set to 15 Å. Default values were used for all other parameters. A further 10 ns MD simulation was submitted using a docking pose which can account for the limited SAR of BIBR1532 and BIBR1591 by employing the program of Desmond, which was developed at D. E. Shaw Research to perform high-speed molecular dynamics simulations of biological systems on conventional commodity clusters. All suggested values of Desmond were employed to run this 10 ns MD simulation. The MD simulation include the following steps: (i) a solvated system for simulation was generated; (ii) positive or negative counter ions were distributed to neutralize the system and additional ions (Naþ and Cl) to were introduced to set the desired ionic strength; (iii) OPLS-AA force field parameters were assigned to the entire molecular system; (iv) the system was relaxed or minimized; (v) the Desmond simulation was run. Compound 3c was docked into the active site of the modeled hTERT-BIBR1532 complex by employing the docking program of €dinger to evaluate its binding mode and binding Glide SP of Schro affinity. 4.9. The measurement of log P The octanol/water partition coefficient is defined as the ratio of a chemical's concentration in the octanol phase to its concentration in the aqueous phase of a two-phase octanol/water system. The logarithm of the ratio of the concentrations of the chemical in the solvents is called log P: the log P value is also known as a measure of lipophilicity. The shake-flask method is a classical and most reliable method of log P determination, which consists of dissolving some of the solute in question in a volume of octanol and water, then measuring the concentration of the solute in each solvent. HPLC method was used to measure the distribution of the solute, then the log P value was calculated by the following formula.
W.-J. Tang et al. / European Journal of Medicinal Chemistry 95 (2015) 166e173
log Poct=wat ¼ log
½soluteoc tan ol ½solutewater
Acknowledgments The authors wish to thank the National Natural Science Foundation of China (No. 21272008, 20802003), Science and Technological Fund of Anhui Province for Outstanding Youth (1408085J04), Anhui Provincial Natural Science Foundation (1308085MH137). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2015.03.039. References [1] T.M. Nakamura, G.B. Morin, K.B. Chapman, S.L. Weinrich, W.H. Andrews, J. Lingner, C.B. Harley, T.R. Cech, Science 277 (1997) 955e959. [2] P. Martínez, M.A. Blasco, Nat. Rev. Cancer 11 (2011) 161e176. [3] S. Sandin, D. Rhodes, Curr. Opin. Struct. Biol. 25 (2014) 104e110. [4] J.W. Shay, S. Bacchetti, Eur. J. Cancer 33 (1997) 787e791. [5] C. Autexier, N. Lue, Annu. Rev. Biochem. 75 (2006) 493e517. [6] X.Q. Wu, C. Huang, X. He, Y.Y. Tian, D.X. Zhou, Y. He, X.H. Liu, J. Li, Cell. Signal. 25 (2013) 2462e2468. [7] J. Ye, V.M. Renault, K. Jamet, E. Gilson, Nat. Rev. Genet. 15 (2014) 491e503. [8] W.C. Hahn, S.A. Stewart, M.W. Brooks, S.G. York, E. Eaton, A. Kurachi, R.L. Beijersbergen, J.H. Knoll, M. Meyerson, R.A. Weinberg, Nat. Med. 5 (1999) 1164e1170.
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