Antiviral Chemistry & Chemotherapy 2010 20:259–268 (doi: 10.3851/IMP1631)
Original article Synthesis and biological evaluation of heat-shock protein 90 inhibitors: geldanamycin derivatives with broad antiviral activities Yan-Ping Li1†, Guang-Zhi Shan1†, Zong-Gen Peng1†, Jian-Hua Zhu1, Shuai Meng1, Tian Zhang1, Lin‑Yan Gao1, Pei-Zhen Tao1, Rong-Mei Gao1, Yu-Huan Li1*, Jian-Dong Jiang1* and Zhuo-Rong Li1* 1
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
*Corresponding author: e-mails:
[email protected];
[email protected];
[email protected] These authors made an equal contribution to this work
†
Background: Previous studies have suggested that geldanamycin (GA) inhibits the replication of several viruses in vitro. Here, we aimed to synthesize and evaluate the antiviral activity of 17-amino-17-demethoxygeldanamycin derivatives. Methods: A series of 17-substituted and 17-,19-disubstituted GA derivatives were screened for antiviral activities against eight different viral strains, including herpesvirus, hepatitis virus, retrovirus and picornavirus. Results: Most of the tested compounds showed inhibitory activity against the viruses and showed reduced cytotoxicity in vitro as compared with the parent compound GA. In vivo efficacy evaluation results showed that
compound 6 noticeably inhibited duckling hepatitis B virus DNA replication in duckling serum after oral administration. Viral rebound did not occur after termination of the treatment. The modified GA derivatives also showed median lethal dose values that were higher than that of the parent GA in mice intraperitoneally treated with the study compounds. Conclusions: Targeting heat-shock protein 90 could be a new antiviral approach that is not prone to the development of drug resistance. The 17-amino-17demethoxygeldanamycin derivatives could be novel agents with potential antiviral activity.
Introduction Viruses with high infection rates and rapid propagation can cause worldwide human and animal disease pandemics. With the emergence of previously unknown viruses and new virus variants in recent years, viral infection remains a major cause of human health problems. Moreover, the existing antiviral agents mainly target the key viral enzymes that are involved in the process of replication. At the same time, many viral enzyme mutations result in drug resistance. Some antiviral agents are also of limited use because of their existing toxic side effects. There is a crucial need for developing new agents with novel antiviral mechanisms and broad antiviral activities. Heat-shock protein 90 (HSP90) is a cellular molecular chaperone that binds to multiple proteins and facilitates the proper refolding of these proteins. It increases the stability of the bound proteins and prevents their degradation through the ubiquitin–proteasome pathway. Previous studies have focused primarily on the ©2010 International Medical Press 1359-6535 (print) 2040-2066 (online)
stabilization of tumour cell oncogenic proteins by HSP90 [1,2]. Recent studies have found that some viral proteins can also directly or indirectly form chaperone complexes with HSP90 to assure stabilization and correct folding; therefore, HSP90 has been identified as an essential host factor required for stabilization of viral proteins. Moreover, attempts to introduce mutations into HSP90 have been difficult because HSP90 is a conserved cellular protein; thus, HSP90 has the potential to be developed as a novel target for broad-spectrum antiviral agents. Optimized HSP90 inhibitors should have broad-spectrum antiviral activity and should be able to reduce the possibility of developing drug resistance. Geldanamycin (GA), an HSP90 inhibitor, is a naturally occurring benzoquinone ansamycin antibiotic that is isolated from the fermentation of Streptomyces hygroscopicus, and has been widely recognized for its potent activity against tumours [3–5]. GA has also been frequently used to investigate the relationship between 259
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HSP90 activity and viral replication. Results from these studies have suggested that GA inhibits the replication of multiple viruses by abolishing the activity of HSP90. For example, by inhibiting HSP90 activity, GA could suppress hepatitis B virus (HBV) DNA replication and RNA packaging because of its specific inhibition of the interaction between the HBV reverse transcriptase and ε RNA [6,7]. GA also suppresses normal functions of viral proteins by preventing the formation of the complex comprising HSP90 and viral proteins; such complexes play an important role in, for example, hepatitis C virus (HCV) RNA replication and in the HIV type-1 (HIV-1) transcription process [8–11]. Inhibition of HSP90 activity also causes inhibition of viral protein synthesis (herpesvirus and some negative-stranded RNA viruses) because it facilitates the degradation of viral RNA polymerases [12–14]. These studies have also indicated that GA could inhibit viral replication by interfering with the chaperone-mediated process in viral protein stabilization and folding. Li et al. [15] and Geller et al. [16] have identified GA as a novel parent compound that exhibits broad-spectrum activities against DNA or RNA viruses in cell cultures and in animals. However, high toxicity and poor oral bioavailability are major drawbacks that limit its use in clinical settings. As previous studies have verified that derivatization of GA by substituting the 17-position of GA with an amino group has greatly decreased the toxicity while retaining the anti-tumour activity [17,18], we synthesized GA derivatives substituted with small molecule alkylamines that consist of heterocycles or strong polar groups, such as phosphates or carbohydrates, in the 17-position to improve both the pharmacokinetic properties and the safety profile of GA. To our knowledge, this is the first report to describe the screening of GA derivatives as antiviral agents. The aim of the present study was to evaluate the antiviral activity of the synthesized 17-amino-17-demethoxygeldanamycin derivatives against a larger panel of viruses, including herpesvirus, hepatitis virus, retrovirus and picornavirus. On the basis of the antiviral activity in vitro, the selected compounds with good efficacy and their structural features were further investigated for their toxicity and antiviral activity in vivo.
Methods Chemistry Unless noted otherwise, all reagents and solvents were purchased from commercial sources and used without purification. GA was purchased from the Shanghai Institute of Pharmaceutical Industry (Shanghai, China) and the purity was >95% by HPLC analysis. 1 H NMR spectra were recorded in CDCl3 or dimethylsulfoxide-d6 (DMSO-d6) solution with a Mercury-400 260
spectrometer (Varian, Inc., Palo Alto, CA, USA. Highresolution mass spectrometry (HRMS) data were obtained using an AutoSpec Ultima-Tof spectrometer (Waters, Milford, PA, USA) with an electrospray ionization (ESI) source. Melting points were determined using an X6 microscope melting point apparatus without correction. Column chromatography was performed on silica gel and eluted with a 1–20% methanol gradient in dichloromethane. All 17-amino-17-demethoxygeldanamycin compounds were synthesized from GA and the corresponding amines (Figure 1). In our previous study, compounds 1, 2, 4, 5, 9 and 10 were synthesized (G-ZS et al., unpublished data). Compounds 17-(piperidinyl-4′yl-methylamino)-17-demethoxygeldanamycin (3), 17-[1′-(2′′,3′′-dihydroxy)propyl]amino-17-demethoxygeldanamycin (6), 17-[2′-(2′′-hydroxyethoxy)ethyl] amino-17-demethoxygeldanamycin (7) and 17-(3′phosphopropyl)amino-17-demethoxygeldanamycin (8) were synthesized using a similar method. The purity of all GA derivatives in this study was >95%, which was achieved using the HLPC area normalization method with a C18 column (5 µm; 4.6×250 mm; Dikma Technologies, Inc., Lake Forest, CA, USA), eluted with water (containing 0.02 mol/l KH2PO4) and methanol at a flow rate of 1 ml/min, and the analyte was detected by ultraviolet absorption at 332 nm. The general procedure for the synthesis of compounds 3, 6, 7 and 8 was as follows: the corresponding amine (0.2 mmol) was added to a solution of GA (0.1 mmol) and dichloromethane (10 ml; methanol or water was added when the amine was not dissolved), and triethylamine (0.23 mmol) was added to release the amine if the amine was in the salt form. The reaction mixture was kept away from light and stirred overnight at room temperature. After concentration in vacuo, the residue was dissolved in ethyl acetate. After washing sequentially with 1 M aqueous hydrochloric acid, water and brine, the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash chromatography on a silica gel, eluted with 1–20% methanol in dichloromethane to yield the product as a purple solid. Compound 3 was synthesized from GA and 4‑aminomethyl piperidine in dichloromethane. Yield: 73%; mp: 140–142°C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 0.63 (d, 3H, J=7.0 Hz), 0.80 (d, 3H, J=7.2 Hz), 0.82–1.08 (m, 4H), 1.24 (m, 2H), 1.50–1.58 (m, 4H), 1.83 (s, 3H), 2.31 (m, 2H), 2.42–2.59 (m, 5H), 2.84–3.29 (m, 7H), 3.38 (d, 1H, J=8.4 Hz), 4.36 (m, 2H), 4.86–4.99 (m, 4H), 5.22 (d, 1H, J=10.0 Hz), 5.41 (t, 1H, J=10.4 Hz), 6.42 (br, 2H), 6.55 (t, 1H, J=11.2 Hz), 6.90–6.96 (m, 3H), 7.12 (d, 1H, J=11.0 ©2010 International Medical Press
Synthesis and antiviral activities of GA derivatives
HRMS (ESI) m/z: 642.2990, calcd for C31H45N3O10Na ([M+Na]+) 690.2997. Compound 7 was synthesized from GA and 2-(2′aminoethoxy)ethanol in mixed solvent of dichloromethane and methanol (3/1, v/v). Yield 92%; mp: 145–147°C. 1H NMR (400 MHz, CDCl3) δ (ppm): 0.98–1.01 (m, 6H), 1.80 (m, 6H), 2.03 (s, 3H), 2.40 (m, 2H), 2.68–2.73 (m, 3H), 3.27 (s, 3H), 3.37 (s, 3H); 3.47–3.49 (m, 1H), 3.57 (m, 1H), 3.63–3.67 (m, 3H), 3.76–3.80 (m, 5H), 4.31 (d, J=10.0 Hz, 2H), 5.20 (s, 1H), 5.84–5.92 (m, 2H), 6.58–6.62 (m, 2H), 6.94 (br, 1H), 6.98 (d, 1H, J=8.4 Hz), 7.21 (s, 1H), 9.17 (br, 1H). HRMS (ESI) m/z: 656.3158, calcd for C32H47N3O10Na ([M+Na]+) 656.3153.
Hz), 7.7 (br, 1H). HRMS (ESI) m/z: 643.3704, calcd for C34H51N4O8 ([M+H]+) 643.3701. Compound 6 was synthesized from GA and 3-amino1,2-propanediol in mixed solvent of dichloromethane and methanol (3/1, v/v). Yield: 61%; mp: 140–142°C. 1 H NMR (400 MHz, CDCl3) δ (ppm): 0.85–1.14 (m, 6H), 1.26–1.28 (m, 2H), 1.52 (s, 1H), 1.64–1.66 (m, 2H), 1.80 (s, 3H), 2.02 (s, 3H), 2.35 (t, J=9.6 Hz, 1H), 2.52–2.58 (m, 2H), 2.73 (br, 2H), 3.26 (s, 3H), 3.35 (s, 3H); 3.43–3.44 (m, 2H), 3.52–3.57 (m, 2H), 3.86 (br, 1H), 4.13 (m, 2H), 4.23–4.26 (m, 1H), 4.30 (d, 1H, J=9.6 Hz), 4.80 (br, 2H), 5.17(s, 1H), 5.79 (t, 1H, J=10.4 Hz), 5.90–5.92 (m, 1H), 6.56 (t, J=11.6 Hz, 1H), 6.95 (d, 1H, J=11.6 Hz), 7.24 (br, 1H).
Figure 1. Synthesis and chemical structures of geldanamycin derivatives 1–10
O H3C
O
R1
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Antiviral Chemistry & Chemotherapy 20.6
NH2
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Compound 8 was synthesized from GA and 3-aminopropylphosphoric acid in the presence of triethylamine in mixed a solvent of dichloromethane, methanol and water (8/3/1, v/v/v). Yield 33%; mp: 143–145°C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 0.81 (d, J=11.2 Hz, 3H), 0.94 (m, 3H), 1.49–1.58 (m, 5H), 1.63 (s, 3H), 1.69–1.79 (m, 2H), 1.83–1.87 (m, 1H), 1.90 (s, 3H), 1.92 (d, J=9.2 Hz, 1H), 2.23–2.29 (m, 2H), 2.44–2.56 (t, J=9.2 Hz, 4H), 3.12–3.40 (m, 6H), 3.47– 3.56 (m, 1H), 4.40 (d, J=7.6 Hz, 1H), 4.98 (s, 1H), 5.52 (d, J=9.6 Hz, 1H), 5.78 (br, 2H), 6.59 (m, 2H), 6.92 (s, 1H), 7.01 (br, 2H), 9.35 (s, 1H), 10.7 (br, 1H). HRMS (ESI) m/z: 690.2772, calcd for C31H46N3O11NaP ([M+Na]+) 690.2762.
Cells African green monkey kidney (Vero) cells, human erythroleukaemia (HEL) cells and metallothionein T-4 (MT-4) cells were obtained from the Institute of Virology, Chinese Academy of Preventive Medicine (Beijing, China). Hepatocellular carcinoma (HepG2.2.15) cells were donated by Mount Sinai Medical Center (New York, NY, USA) and human liver cells (Huh7.5) were kindly provided by Vertex Pharmaceuticals, Inc., (Boston, MA, USA).
Viruses and plasmids Human cytomegalovirus (HCMV; AD169 strain), herpes simplex virus type-1 (HSV-1; KOS strain), HSV type-2 (HSV-2; SAV strain), HIV-1 (strain IIIB) and Coxsackie viruses (Coxsackie virus B3 Nancy strain [Cox B3] and Coxsackie virus B6 Schmitt strain [Cox B6]) were all from the Institute of Virology, Chinese Academy of Preventive Medicine. The plasmid pFL-J6/JFH/JC1 with the complete chimeric HCV complementary DNA was provided by Vertex Pharmaceuticals, Inc. HCV virus stock was prepared as previously reported [19].
Reagents and animals Lamivudine (3TC) was purchased from GlaxoSmith Kline (Suzhou, China), zidovudine was purchased from Shanghai Desano Co. (Shanghai, China), aciclovir was purchased from Hubei Yitai Co. (Tianmen, China) and ribavirin was purchased from Xinxiang Chemial Pharmaceutical Co. (Xinxiang, China). Interferon-α2b was from Schering–Plough Co. (Brinny, Ireland). We synthesized valganciclovir from ganciclovir; the purity was >95% by HPLC analysis. Kunming mice were purchased from the Chinese Academy of Medical Sciences (Beijing, China). Beijing ducklings were purchased from Beijing Nanyan Fowl, Inc. (Beijing, China). The animals were housed in standard laboratories at 22 ±1°C and had free access to food and water except for a short fasting period before intraperitoneal or oral drug administration. These 262
experiments were approved by our institution’s animal Ethics Committee (Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China).
Cytotoxicity assays The cytotoxicity of tested compounds in Vero, HEL and HepG2.2.15 cells were monitored using cytopathic effect (CPE) assays as described previously [20]. Vero and HEL cells (2.5×104 cells/well) were cultured in 96-well plates. After 24 h, the monolayers were incubated in the presence of various concentrations of the test compounds. After 3 days of culture at 37°C and 5% CO2, the cells were assayed by CPE. HepG2.2.15 cells (2.0×104 cells/ well) were cultured in 96-well plates. After 24 h, the monolayer cells were incubated in the presence of various concentrations of the test compounds for 4 days, then washed and a fresh medium containing the test compounds was added. After 8 days of incubation at 37°C and 5% CO2, the cells were assayed by CPE. The 50% cytotoxic concentration (CC50) was defined as the compound concentration that reduced the viability of untreated cell cultures by 50%. The cytotoxicity in MT-4 and Huh7.5 cells were monitored by tetrazolium (MTT) assays [15]. Huh7.5 cells (1×104 cells/well) were planted into 96-well plates. After 6 h, test compounds with various concentrations were added. Cell viability was detected using MTT assays at 96 h. MT-4 cells (2×104 cells/well) were plated into a 96-well plate and incubated in the presence of various concentrations of the test compounds. After 4 days of culture at 37°C and 5% CO2, the cell viability was quantified using MTT assays.
Anti-HBV activity assay in vitro The anti-HBV activities of compounds together with a positive control drug were tested in HepG2.2.15 cells. The HepG2.2.15 cells were cultured in 96-well plates and treated with the test compounds at 37°C for 4 days, then washed and a fresh medium containing the test compounds was added. After 8 days of incubation at 37°C and 5% CO2, the cells were harvested and intracellular DNA was extracted. The inhibition of the viral DNA in treated cells versus untreated cells was determined by dot blot hybridization.
Anti-HCV activity assay in vitro Huh7.5 cells were cultured in 96-well plates at a density of 1×104 cells/well. After 6 h of incubation, cells were infected with HCV virus stock (45 IU/cell) and treated concomitantly with tested compounds or interferonα2b. At 96 h after inoculation in 5% CO2 at 37°C, cells were harvested and intracellular total RNA was extracted using an RNeasy mini kit (Qiagen, Hilden, Germany). The HCV RNA was quantified directly ©2010 International Medical Press
Synthesis and antiviral activities of GA derivatives
with a one-step real-time reverse transciptase PCR kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol.
Anti-HIV-1 activity assay in vitro The anti-HIV-1 activity of the test compounds on HIV-1 replication was measured by inhibition of HIV-1 p24 antigen in MT-4 cell cultures as described previously [21]. MT-4 cells (2×105 cells/ml) infected with HIV-1 at 100 median tissue culture infective dose (TCID50)/ ml were cultured in 96-well plates and incubated in the presence of various concentrations of test compounds or a positive control drug. After 4 days of culture at 37°C and 5% CO2, the culture supernatants were collected and assayed for their p24 antigen levels by ELISA.
Anti-HSV, Coxsackie virus and HCMV activity assays in vitro Confluent Vero cells grown in 96-well microplates were infected with 100 TCID50 HSV-1, HSV-2, Cox B3 or Cox B6 virus. HEL cells were infected with HCMV. After 1 h adsorption at 37°C, the monolayer cells were washed with phosphate-buffered saline and incubated at 37°C in the maintenance medium (minimum essential medium plus 2% fetal bovine serum) with or without different concentrations of the test compounds or a positive control drug. The CPE in cells was recorded when the CPE of the viral control group reached >75% and the antiviral activity of compound was determined using Reed–Muench analysis [22].
Acute toxicity tests in mice Kunming mice (20 ±2 g) were divided into groups with eight mice per group. The control group received DMSO only. On the basis of the pre-experiment results, each of the test compounds dissolved in DMSO was administrated with three doses to three groups of animals. Mice were placed unrestrained in their cages after treatment and observed for 5 days to monitor behavioural changes and death. The median lethal dose (LD50) was calculated using the Bliss method according the death number.
Anti-DHBV DNA activity in a DHBV infection model One-day-old Beijing ducklings, inoculated intravenously with duckling hepatitis B virus (DHBV)-DNApositive serum from Shanghai ducks (0.2 ml/duckling), were randomly divided into four groups (6 ducklings/ group) for treatment. The virus control group was treated with 0.1% carboxymethylcellulose in water, the positive drug group was treated with 3TC (50 mg/kg) and the two test compound groups were treated with compound 1 (30 mg/kg) or compound 6 (30 mg/kg). Oral administration was started at day 7 after infection and was carried out twice daily for 10 days. Blood Antiviral Chemistry & Chemotherapy 20.6
samples were taken and serum samples were prepared for analysis of DHBV DNA levels before treatment (T0), on days 5 (T5) and 10 (T10) during treatment and on day 3 (P3) after termination of treatment. The detection of DHBV DNA levels in the serum samples were done using the dot blot hybridization method as described previously [23,24]. Briefly, 50 µl of duckling serum was spotted directly onto the nitrocellulose filters. After denaturation and fixation, the filters were hybridized with a DHBV 32PDNA probe. Phosphorimaging dots were obtained on the films using autoradiography. Films were then scanned for the integrated optical density (IOD) of each dot and analysed using Gel-Pro Analyzer 3.1 (Media Cybernetics, Inc., Silver Spring, MD, USA). The IOD values of imaging dots were displayed as a marker of DHBV DNA levels. In order to eliminate the background difference between groups, the relative ratio (IOD RR) between the post- and pretreatment result of each group was used to show the change in DHBV DNA levels. Lower IOD RR values represent a greater decrease in DHBV DNA levels.
Statistical analyses The repeated measurement data from in vivo anti-DHBV DNA activity tests were analysed using the general linear models procedure in SPSS version 13.0 (SPSS, Inc., Chicago, IL, USA) for Windows. Multivariate analysis of variance was used to test the significance of the group effect. The criterion for significance was set at P31.11 0.776 2.032 26.90 14.17 17.29 0.733 274.3 0.037
0.006 0.091 0.030 3.453 0.213 0.095 1.000 0.142 3.324 0.158 23.88 0.0006
17,913 86 18 >9 3 21 26 99 5 4 11 61
>222 44.74 29.94 >31.11 19.92 20.45 170.7 175.0 >29.95 106.2 >274.3 21.74
16.42 2.003 1.461 27.62 1.598 1.492 13.29 8.457 3.070 1.753 158.4 0.499
>12 22 20 >1 12 13 12 20 >10 60 >1 43
30.81 1.745 2.862 >31.11 4.932 1.476 21.54 8.883 10.18 1.681 190.2 0.891
>14 25 10 –b 4 13 7 19 >3 63 >1 24
a The positive control drugs were valganciclovir or aciclovir for anti-human cytomegalovirus (HCMV) and anti-herpes simplex virus (HSV) tests, respectively. bThe selectivity index (SI) could not be determined under the test conditions. CC50, 50% cytotoxic concentration; GA, geldanamycin; HEL, human erythroleukaemia cells; HSV-1, herpes simplex virus type-1; HSV-2, herpes simplex virus type-2; IC50, 50% inhibition concentration; Vero, African green monkey kidney cells.
Table 2. Anti-HBV and anti-HCV activity and cytotoxicity of GA derivatives Compound
HepG2.2.15 CC50, µM
HBV IC50, µM
SI
Huh7.5 CC50, µM
HCV IC50, µM
SI
Positive druga 1 2 3 4 5 6 7 8 9 10 GA
5,103 2.428 0.167 27.83 26.30 0.762 47.20 9.073 8.611 24.79 91.47 7.634
3.882 0.136 0.123 NA NA NA 2.598 1.309 NA 1.250 NA 0.123
1,314 17 –b –b –b –b 18 6.9 –b 19 –b 62
>6,000 0.910 0.380 7.848 0.768 0.222 1.533 0.733 0.726 0.237 179.5 0.053
0.52 0.531 NA 6.145 NA 0.103 NA 0.449 NA NA NA NA
>11,538 1 –b –b –b 2 –b 1 –b –b –b –b
a The positive control drug was lamivudine or interferon-α2b for hepatitis B virus (HBV) or hepatitis C virus (HCV), respectively. bThe selectivity index (SI) could not be determined under the test conditions. CC50, 50% cytotoxic concentration; GA, geldanamycin; HepG2.2.15, hepatocellular carcinoma cells; Huh7.5, human liver cells; IC50, 50% inhibition concentration; NA, not active under test condition.
As summarized in Table 1, the CC50 values of the tested compounds were all significantly higher than that of GA, both in HEL and Vero cells: the CC50 values of all GA derivatives in the HEL cells were >10-fold of that of GA (0.037 µM). Among the compounds, the cytotoxicity of compounds 6, 7 and 10 showed a significant decrease in cell cultures, and compound 10 (with disubstitutions at the 17- and 19-positions of GA) showed no toxicity even at the highest test concentration (274 µM) used in the cell lines. These data indicated that modifying GA with an amino group in the 17-position could decrease the cytotoxicity of GA derivatives in cell culture.
In vitro hepatitis virus inhibitory activities The anti-HBV activity of GA and its derivatives was tested in HepG2.2.15 cells (Table 2). Compounds 1, 2, 6, 7 and 9 showed inhibitory activities on HBV 264
replication under test concentrations, and compounds 1 and 2 showed IC50 values similar to that of GA. Compounds 3, 4, 5, 8 and 10 were not active against HBV. The anti-HCV activity of GA and its derivatives was tested in Huh7.5 cells. The CC50 values of all GA derivatives were higher than that of GA (Table 2), indicating that the cytotoxicity of the GA derivatives were lower than that of GA; however, the derivatives showed no significant anti-HCV activity under the test concentration.
In vitro HIV-1 virus inhibitory activities Anti-HIV-1 activity of compounds 1–5 were tested in MT-4 cells. The results are shown in Table 3. All of the compounds inhibited HIV-1 replication. Compounds 1, 2 and 4 possessed inhibitory activity that was stronger than that of GA. Higher SI values of GA derivatives over GA were also obtained in MT-4 cells. ©2010 International Medical Press
Synthesis and antiviral activities of GA derivatives
Table 3. Anti-HIV-1 activity and cytotoxicity of GA derivatives Index
AZT
1
2
3
4
5
GA
CC50, µM IC50, µM SI
2.181 0.001 2173
4.280 282
1.537 101
1.197 0.093 12
1.233 0.013 90
3.668 0.222 16
0.552 0.099 5
AZT, zidovudine; CC50, 50% cytotoxic concentration; GA, geldanamycin; IC50, 50% inhibition concentration; SI, selectivity index.
Table 4. Anti-Coxsackie virus activity and cytotoxicity of GA derivatives Compound
Vero CC50, µM
Cox B3 IC50, µM
SI
Cox B6 IC50, µM
SI
Ribavirin 1 2 3 4 5 6 7 8 9 10 GA
>6,142 >30.35 >30.45 71.91 17.61 19.06 107.6 105.2 61.75 >28.74 158.4 21.41
1339 3.840 1.476 26.84 0.494 0.231 27.84 15.05 2.876 4.786 >30.48 0.155
>4 >7 >20 2 35 82 3.86 6.99 21.47 >6 –a 137
1,958 1.442 5.861 3.843 1.446 1.175 8.342 2.019 9.989 >1.179 >30.48 0.035
>3.1 >21 >5 18 12 16 12 52 6 –a –a 611
The selectivity index (SI) could not be determined under the test conditions. CC50, 50% cell cytotoxic concentration; Cox B3, Coxsackie virus B3 Nancy strain; Cox B6, Coxsackie virus B6 Schmitt strain; GA, geldanamycin; IC50, 50% inhibition concentration; SI, selectivity index; Vero, African green monkey kidney cells.
a
The lack of effective antiviral drugs against picornavirus led us to investigate the anti-Coxsackie virus activity of these GA derivatives (Table 4). Except for compounds 4 and 5, the others had lower toxicity than that of GA in Vero cells. Although none of derivatives was more potent than GA according to the IC50 and SI values for either Cox B3 or Cox B6 virus, most compounds were superior to the positive control drug, ribavirin, in terms of antiviral efficacy. Ribavirin is currently used for clinical therapy against Coxsackie viral infection.
Acute toxicity in mice To further evaluate the toxicity of GA derivatives, we investigated the acute toxicity of compounds 1, 4, 5, 6 and 9 in mice after a single dose by intraperitoneal administration. The results were compared with that of GA. In our study, mice often died 3 days after drug injection; otherwise, mice recovered to a normal state within 5 days. The LD50 of tested compounds are shown in Figure 2. Among these compounds, GA was shown to be the most toxic with an LD50 value of 17.25 mg/kg. The LD50 values of compounds 1, 4, 5, 6 and 9 were 295.4, 122.8, 238.1, 203.8 and 35.48 mg/kg, respectively, indicating that all of the test compounds had lower toxicity than that of GA in mice. This was in agreement with the results from cytotoxicity assay. Antiviral Chemistry & Chemotherapy 20.6
Figure 2. The acute toxicity of GA derivatives in mice
LD50, mg/kg
In vitro Coxsackie virus inhibitory activities
350 300 250 200 150 100 50 0 GA
1
4 5 Compound
6
9
GA, geldanamycin; LD50, median lethal dose.
In vivo inhibitory activity of DHBV DNA replication in a DHBV infectious model Based on the in vitro anti-HBV activity results and the relationships between chemical structure and antiviral activity as well as toxicity, compounds 1 and 6 were chosen for further evaluation of their in vivo efficacy against DHBV DNA replication. GA derivatives were administrated orally to ducklings. Multivariate analyses of variance indicated that 3TC and compound 6 significantly decreased DHBV 265
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HSP90 inhibitor for treatment of tumours and has been enrolled into multiple clinic trials [25]. In our study, the majority of tested compounds also appeared to be less cytotoxic than GA in the cell cultures; the LD50 values of the selected six compounds were higher than that of GA in mice, giving further evidence of the low toxicity of these derivatives. We also found that the 17,19-disubstituted derivative (10) showed the greatest decrease in toxicity; however, it should be noted that the antiviral activity of compound 10 almost disappeared in the anti-Coxsackie and hepatitis virus assays, which suggests that the 19-substituent modification is not an ideal choice in terms of antiviral activity. The introduction of a group at the 19-position of GA might interfere with the binding of GA derivatives to HSP90 because of steric effects. 17-Substituted GA derivatives contained both stronger and weaker antiviral activity than GA, but all had a largely decreased toxicity. According to the crystal structure of the GA–HSP90 complex [26], the substituent in the 17-position of GA is exposed to the outside cavity of the HSP90 protein, which suggests that the difference of substituents at the 17-position of GA are not crucial for the interaction of the test compounds with the HSP90 protein. This observation also helps us to understand why there was no obvious regularity observed between antiviral activity and structural difference in the 17-position in this study. Noticeably, compounds 1, 2, 6, 7 and 9 demonstrated a potent antiviral activity against at least four of the eight tested viral strains. This led us to speculate that
DNA levels in serum samples, it also revealed a greater decrease in DHBV DNA levels at T10 in both the 3TC (P
