Accepted Manuscript Synthesis, in vitro antimicrobial and cytotoxic activities of new carbazole derivatives of ursolic acid Wen Gu, Yun Hao, Guang Zhang, Shi-Fa Wang, Ting-Ting Miao, Kang-Ping Zhang PII: DOI: Reference:
S0960-894X(14)01318-3 http://dx.doi.org/10.1016/j.bmcl.2014.12.021 BMCL 22266
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
24 September 2014 7 December 2014 9 December 2014
Please cite this article as: Gu, W., Hao, Y., Zhang, G., Wang, S-F., Miao, T-T., Zhang, K-P., Synthesis, in vitro antimicrobial and cytotoxic activities of new carbazole derivatives of ursolic acid, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.12.021
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Synthesis, in vitro antimicrobial and cytotoxic activities of new carbazole derivatives of ursolic acid Wen Gu a,∗, Yun Hao a, Guang Zhang b, Shi-Fa Wang a, Ting-Ting Miao a, Kang-Ping Zhang a
a
Jiangsu Key Lab of Biomass-based Green Fuels and Chemicals, College of Chemical Engineering, Nanjing
Forestry University, Nanjing 210037, P. R. China b
Jiangsu Key Lab of Molecular Medicine, State Key Lab of Pharmaceutical Biotechnology, Medical School,
Nanjing University, Nanjing 210093, P. R. China
∗
Corresponding author. Tel.: +86 25 8542 8369; fax: +86 25 8542 7396. E-mail address: [email protected] (W. Gu).
Abstract A series of new carbazole derivatives of ursolic acid were designed and synthesized in an attempt to develop potent antimicrobial or antitumor agents. Their structures were confirmed by using IR, HRMS and 1H NMR analysis. All the synthesized compounds were evaluated for their antimicrobial activity against four bacterial and three fungal strains using serial dilution method. Compounds 3a, 3b, 4a, 4b and 5a-f exhibited significant antibacterial activity against at least one tested bacteria with MIC values of 3.9-15.6 µg/ml. In addition, the in vitro cytotoxicity of these compounds were also assayed against two human tumor cell lines (SMMC-7721 and HepG2) using MTT colorimetric method. From the results, compounds 5a-e and 5h displayed pronounced cytotoxic activity with IC50 values below 10 µM. Specially, compound 5e was found to be the most potent compound with IC50 values of 1.08 ± 0.22 and 1.26 ± 0.17µM against SMMC-7721 and HepG2 cells, respectively, comparable to those of doxorubicin. In addition, compound 5e showed reduced cytotoxicity against noncancerous LO2 cells with IC50 value of 5.75 ± 0.48 µM. Keywords: Ursolic acid, Carbazole, Synthesis, Antimicrobial activity, Cytotoxic activity
Triterpenoids are a large and diverse class of natural products widely distributed in the plant kingdom. It is generally believed that one of the physiological functions of triterpenoids is to enhance the resistance of plants against the invasion of plant pathogens.1 Triterpenoids exhibit a broad spectrum of pharmacological activities which are under active investigations worldwide for various therapeutic properties.2-4 Ursolic acid (UA, 3β-hydroxy-urs-12-en-28-oic acid, 1) is a well-known ursane-type pentacyclic triterpenoid that has been reported to possess a wide range of biological activities, including antimicrobial,5 antitumor,2,6 antiviral,7 antiprotozoal,8 antioxidant,9 anti-inflammatory,10 antidiabetic11 as well as anti-osteoporosic activities.12 However, the low bioavailability of UA in vivo restricts its clinical application.13 Therefore, structural modifications in UA have been widely investigated in recent years to improve its biological activities and bioavailability.14,15 Carbazole is an important type of nitrogen-containing aromatic heterocyclic motif found in many natural and synthetic molecules with various pharmacological activities including antimicrobial, anticancer, anti-inflammatory, antiviral, antioxidant activities, etc.16-20 Ellipticine, as a typical example, is a natural carbazole alkaloid exhibiting powerful antiproliferative properties and has been used clinically as an anticancer drug. A number of ellipticine derivatives have also been designed and synthesized with improved anticancer activities against a panel of cancer cell lines.21-23 Thus it is speculated that the fusion of a carbazole moiety with the molecule of UA would probably change its physicochemical properties and lead to better biological activities. On the other hand, previous studies indicated that the incorporation of polar moiety onto the C-3 or C-28 position might improve the water solubility and thus clinical utility.24,25 In a former literature, UA derivatives were conjugated with different amino acids on the C-28 carboxyl groups. Some
derivatives with amino groups (positively charged) exhibited more potent cytotoxic activity than UA and the derivatives bearing carboxyl groups (negatively charged).26 Therefore, the introduction of amine moieties onto C-28 carboxyl groups of UA derivatives might also be benefical to their biological activities. Prompted by the above facts and in continuation with our efforts towards potential natural product-derived antimicrobial and anticancer agents, a series of novel carbazole derivatives of UA were designed and synthesized. The reaction sequence employed in the synthesis of the target compounds was shown in Scheme 1. First, the starting material UA (1) was oxidized by Jone’s reagent to afford 3-oxo-ursolic acid (2) in 76% yield. Subsequently, a series of carbazole derivatives of UA (3a-h) were synthesized from compound 2 and different substituted phenylhydrazine hydrochlorides in 55-65% yields through Fisher indole reactions. Further the esterification of the carboxyl groups of compounds 3a-h proceeded under known procedure to afford the corresponding ester products 4a-h in 82-89% yields. On the other hand, compounds 3a-h were converted to the corresponding amide derivatives 5a-h in 73-85% yields by reacting with 3-(dimethylamino)-1-propylamine (DMAPA) in the presence of 1-hydroxybenzotriazole (HOBt) and dicyclohexylcarbodiimide (DCC). All the synthesized target compounds were purified by recrystallization or silica gel column chromatography, and the structures of these compounds were characterized by their HR-MS, IR and 1H NMR spectra. Next, all the synthesized compounds were screened for the antimicrobial activity. The microorganisms used in the study were two Gram-positive bacteria (Bacillus subtilis CGMCC 1.1162, Staphylococcus aureus CGMCC 1.1361), two Gram-negative bacteria (Escherichia coli CGMCC 1.1571, Pseudomonas fluorescens CGMCC 1.1828) and three fungi (Candida albicans
CGMCC 2.2086, Candida tropicalis CGMCC 2.3967 and Aspergillus niger CGMCC 3.316). The minimum inhibitory concentrations (MICs) of the target compounds were determined through a modified broth microdilution method.27 The widely used antibacterial drug amikacin sulfate and antimycotic drug ketoconazole were co-assayed as positive controls against the tested bacteria and fungi, respectively. The results of the antimicrobial assay were shown in Table 1. As illustrated in Table 1, most of the synthesized compounds exhibited inhibitory activity against both Gram-positive and Gram-negative bacteria with MIC values < 100 µg/ml. Among them, compounds 3a, 3b, 4a, 4b and 5a-f displayed considerable antibacterial activity against at least one tested bacteria with MIC values ranging from 3.9 to 15.6 µg/ml. Specially, compound 5b exerted comparatively the highest inhibitory effect against B. subtilis with MIC value of 3.9 µg/ml, near to that of positive control. The compound also showed significant inhibition (MIC: 7.8 µg/ml) against the other three bacterial strains. In addition, compounds 5a and 5c also registered same inhibitory potency (MIC: 7.8 µg/ml) against B. subtilis and S. aureus, respectively. On the other hand, compounds 3c-h, 4c-h, 5g and 5h were found to display moderate, weak or even no inhibition against the four bacterial strains. Concerning the antifungal activity, most of the synthesized compounds showed mild or no inhibition against the three tested fungi. Among them, compounds 3d, 4e, 5a, 5b, 5c and 5f exhibited moderate inhibitory activity (MIC: 31.2 µg/ml) against A. niger. In addition, 5a and 5b were also inhibitory to C. albicans and/or C. tropicalis at the same concentration. From the in vitro antimicrobial activity data, preliminary structure-activity relationship of the synthesized compounds was studied. A number of derivatives showed enhanced activities than the parent intermediate 2, which indicated that the incorporation of carbazole moiety to the molecule
was beneficial to the antimicrobial activity. The results also suggested that the substituents at C-28 position of these derivatives had an important effect on their antimicrobial activity. In general, the derivatives with N-(dimethylamino)propyl amide side chains (5a-h) exhibited higher antibacterial activity than other derivatives with carboxyl groups (3a-h) and methyl ester groups (4a-h). For example, compound 5b exhibited significant activity against four tested bacteria (MIC: 3.9 ~ 7.8 µg/ml), while its analogs 3b and 4b were only moderately inhibitory to the four strains (MIC: 15.6 ~ 31.2 µg/ml). Moreover, the antimicrobial activity of these UA derivatives were also substantially influenced by the substituents on the phenyl rings. Among derivatives 5a-h, compounds 5b and 5c with electron-withdrawing substituents (F and Cl) were more potent against tested bacteria than those with electron-donating substituents (CH3, OCH3 and OC2H5). However, compound 5d with relatively weak electron-withdrawing substituent (Br) only exhibited moderate antimicrobial activity. Similar results could be observed in derivatives 3a-h and 4a-h. The results indicated that the appropriate electron density on the phenyl ring of these derivatives might be closely related to high antimicrobial activity. The target compounds were also evaluated for their in vitro cytotoxicity against two human hepatocarcinoma cell lines (SMMC-7721 and HepG2) using MTT assay method.28 The proverbial anticancer drug doxorubicin was used as the positive control. The results of the test compounds expressed as IC50 values (concentration required to inhibit tumor cell proliferation by 50%) are presented in Table 2. From the results, these UA derivatives exhibited varying degrees of inhibitory activity against the two cell lines. Compounds 5a-e and 5h showed significant cytotoxic activity with IC50 values below 10 µM. Notably, compound 5e was the most potent compound in the series with IC50 values of 1.08 ± 0.22 and 1.26 ± 0.17 µM against SMMC-7721 and HepG2
cells, respectively, which were comparable to those of positive control doxorubicin. In addition, compound 5e was also assayed for the in vitro cytotoxic activity against the noncancerous human hepatocyte cell line LO2, which showed reduced cytotoxicity with IC50 value of 5.75 ± 0.48 µM. Compounds 3a-f, 3h and 5g also showed moderate cytotoxicity against one or two cancer cell lines. However, nearly all of compounds 4a-h were inactive to tested cancer cells with IC50 values > 100 µM. The aforementioned results suggested that the cytotoxic activity of these derivatives was also markedly influenced by their structural properties. Similar to the antimicrobial activity, the cytotoxic activity of amide derivatives 5a-h was found to be much more potent than the other two series of derivatives, indicating that the N-(dimethylamino)propyl moiety was crucial for their cytotoxic activity. Contrarily, the esterification of the C-28 carboxyl group would substantially decrease the cytotoxicity of the derivatives. A possible explanation might be the electrostatic interaction between the derivatives and the cancer cells. The outer surface of assayed cancer cells presented net negative charge in physiological environment, which facilitated the accumulation of positively charged compounds around cells.26,29,30 Therefore, compounds 5a-h with amine moiety (positively charged) would obtain higher local drug concentration and enhanced cytotoxic activity. On the other hand, the substituents on the benzene rings of these derivatives affected the cytotoxic activity to a certain extent. For compounds 5a-h, derivatives with relatively more lipophilic substituents such as H atom (5a), halogen atoms (5b-d), CH3 (5e) and OC2H5 (5h) exhibited stronger inhibitory activity than those with more polar methoxy groups (5f and 5g). The derivatives 3a-h displayed similar results as well. The introduction of substituents with higher lipophilicity on the indole benzene ring would probably lead to better membrane permeability, and
thus increased their pharmaceutical effects.31,32 As for methoxy groups, the relocation of the methoxy group from the p-position (3f and 5f) to the o-position (3g and 5g) decreased the cytotoxic activity remarkably, which suggested that p-substituted methoxy substituent was more favorable to the cytotoxicity than o-substituted methoxy group. In summary, a series of novel carbazole derivatives of ursolic acid were designed, synthesized, and evaluated for their in vitro antimicrobial and cytotoxic activities. As a result, a number of derivatives exhibited promising antimicrobial and/or cytotoxic activities when compared with the positive
controls.
Among
them,
compound
5b
with
5-fluoroindole
moiety
and
N-(dimethylamino)propyl amide side chain showed pronounced antibacterial activity against the tested bacteria. Moreover, its analog 5e with 5-methylindole moiety displayed potent cytotoxic activity against two human hepatocarcinoma cell lines comparable to doxorubicin. The activities of these derivatives were closely relevant to their structural properties. These findings would open a new avenue for the development of this class of UA derivatives as prospective antimicrobial or anticancer agents. Further investigations, including extensive structural modifications and in-depth SAR analysis, cytotoxic assay against other tumor cell lines and pharmacological studies are to be performed for discovering new derivatives with better activities and revealing their exact mechanism of action.
Acknowledgements The authors are grateful to the National Natural Science Foundation of China for financial support (Project: 31000273). This work was also supported by a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). The authors
would like to thank the Advanced Analysis & Testing Center of Nanjing Forestry University for the measurements of NMR data.
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://
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Table 1 Antimicrobial activity of synthesized compounds (MIC values are in µg/ml) Compd No.
Antibacterial activity
Antifungal activity
Gram positive bacteria
Gram negative bacteria
B. subtilis
S. aureus
E. coli
P. fluorescens
A. niger
C. albicans
C. tropicalis
2
31.2
62.5
31.2
62.5
>100
>100
>100
3a
15.6
31.2
15.6
15.6
>100
>100
>100
3b
15.6
15.6
15.6
31.2
62.5
62.5
>100
3c
31.2
31.2
31.2
31.2
>100
>100
>100
3d
31.2
62.5
62.5
62.5
31.2
62.5
62.5
3e
62.5
62.5
31.2
62.5
>100
>100
>100
3f
31.2
31.2
31.2
62.5
>100
>100
62.5
3g
62.5
62.5
62.5
62.5
>100
>100
>100
3h
62.5
62.5
62.5
62.5
>100
>100
>100
4a
31.2
15.6
31.2
62.5
62.5
62.5
62.5
4b
31.2
15.6
31.2
31.2
62.5
62.5
62.5
4c
31.2
62.5
>100
62.5
62.5
>100
>100
4d
62.5
31.2
>100
62.5
>100
>100
>100
4e
31.2
31.2
>100
62.5
31.2
>100
62.5
4f
31.2
31.2
62.5
>100
>100
>100
>100
4g
31.2
31.2
>100
>100
>100
>100
>100
4h
31.2
31.2
>100
62.5
>100
>100
>100
5a
7.8
15.6
15.6
31.2
31.2
31.2
62.5
5b
3.9
7.8
7.8
7.8
31.2
31.2
31.2
5c
15.6
7.8
15.6
31.2
31.2
62.5
62.5
5d
31.2
31.2
31.2
15.6
62.5
>100
62.5
5e
15.6
31.2
31.2
31.2
62.5
>100
>100
5f
15.6
31.2
31.2
31.2
31.2
62.5
62.5
5g
31.2
62.5
31.2
31.2
>100
>100
>100
5h
31.2
62.5
31.2
62.5
>100
>100
>100
Amikacin
0.9
0.9
1.9
0.9
−
−
−
Ketoconazole
−
−
−
−
7.8
3.9
3.9
Table 2 IC50 values of the synthesized compounds against two hepatocarcinoma cell lines. Compound
IC50 value (µM) SMMC-7721
HepG2
2
>100
>100
3a
40.36 ± 1.72
25.77 ± 0.83
3b
16.31 ± 0.52
15.21 ± 0.90
3c
29.88 ± 1.43
30.95 ± 2.78
3d
62.63 ± 2.89
38.40 ± 1.86
3e
13.80 ± 0.85
17.03 ± 2.27
3f
31.73 ± 0.98
34.60 ± 0.77
3g
>100
>100
3h
24.48 ± 1.58
33.05 ± 2.24
4a
>100
>100
4b
>100
97.00 ± 2.02
4c
>100
>100
4d
>100
>100
4e
>100
>100
4f
>100
>100
4g
>100
>100
4h
>100
>100
5a
2.12 ± 0.35
2.78 ± 0.21
5b
4.60 ± 1.17
6.03 ± 1.12
5c
2.78 ± 0.31
4.02 ± 0.86
5d
6.80 ± 1.09
5.65 ± 1.43
5e
1.08 ± 0.22
1.26 ± 0.17
5f
12.77 ± 0.53
11.37 ± 0.69
5g
36.18 ± 2.01
88.78 ± 3.98
5h
4.88 ± 0.72
6.25 ± 1.37
Doxorubicin
0.62 ± 0.16
0.77 ± 0.12
Legend Scheme 1. Synthetic route for the carbazole derivatives (3a-h, 4a-h and 5a-h) from ursolic acid. Reagents and conditions: (a) Jone’s reagent, acetone, 0 °C, 5 h; (b) substituted phenylhydrazine hydrochloride, EtOH, conc. HCl, reflux for 3h; (c) i. SOCl2, benzene, reflux for 3h; ii. Methanol, reflux for 2h; (d) DMAPA, HOBt, DCC, CH2Cl2, rt, 12h.
Scheme 1.
Graphic Abstract
S0960-894X(14)01318-3 http://dx.doi.org/10.1016/j.bmcl.2014.12.021 BMCL 22266
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
24 September 2014 7 December 2014 9 December 2014
Please cite this article as: Gu, W., Hao, Y., Zhang, G., Wang, S-F., Miao, T-T., Zhang, K-P., Synthesis, in vitro antimicrobial and cytotoxic activities of new carbazole derivatives of ursolic acid, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.12.021
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Synthesis, in vitro antimicrobial and cytotoxic activities of new carbazole derivatives of ursolic acid Wen Gu a,∗, Yun Hao a, Guang Zhang b, Shi-Fa Wang a, Ting-Ting Miao a, Kang-Ping Zhang a
a
Jiangsu Key Lab of Biomass-based Green Fuels and Chemicals, College of Chemical Engineering, Nanjing
Forestry University, Nanjing 210037, P. R. China b
Jiangsu Key Lab of Molecular Medicine, State Key Lab of Pharmaceutical Biotechnology, Medical School,
Nanjing University, Nanjing 210093, P. R. China
∗
Corresponding author. Tel.: +86 25 8542 8369; fax: +86 25 8542 7396. E-mail address: [email protected] (W. Gu).
Abstract A series of new carbazole derivatives of ursolic acid were designed and synthesized in an attempt to develop potent antimicrobial or antitumor agents. Their structures were confirmed by using IR, HRMS and 1H NMR analysis. All the synthesized compounds were evaluated for their antimicrobial activity against four bacterial and three fungal strains using serial dilution method. Compounds 3a, 3b, 4a, 4b and 5a-f exhibited significant antibacterial activity against at least one tested bacteria with MIC values of 3.9-15.6 µg/ml. In addition, the in vitro cytotoxicity of these compounds were also assayed against two human tumor cell lines (SMMC-7721 and HepG2) using MTT colorimetric method. From the results, compounds 5a-e and 5h displayed pronounced cytotoxic activity with IC50 values below 10 µM. Specially, compound 5e was found to be the most potent compound with IC50 values of 1.08 ± 0.22 and 1.26 ± 0.17µM against SMMC-7721 and HepG2 cells, respectively, comparable to those of doxorubicin. In addition, compound 5e showed reduced cytotoxicity against noncancerous LO2 cells with IC50 value of 5.75 ± 0.48 µM. Keywords: Ursolic acid, Carbazole, Synthesis, Antimicrobial activity, Cytotoxic activity
Triterpenoids are a large and diverse class of natural products widely distributed in the plant kingdom. It is generally believed that one of the physiological functions of triterpenoids is to enhance the resistance of plants against the invasion of plant pathogens.1 Triterpenoids exhibit a broad spectrum of pharmacological activities which are under active investigations worldwide for various therapeutic properties.2-4 Ursolic acid (UA, 3β-hydroxy-urs-12-en-28-oic acid, 1) is a well-known ursane-type pentacyclic triterpenoid that has been reported to possess a wide range of biological activities, including antimicrobial,5 antitumor,2,6 antiviral,7 antiprotozoal,8 antioxidant,9 anti-inflammatory,10 antidiabetic11 as well as anti-osteoporosic activities.12 However, the low bioavailability of UA in vivo restricts its clinical application.13 Therefore, structural modifications in UA have been widely investigated in recent years to improve its biological activities and bioavailability.14,15 Carbazole is an important type of nitrogen-containing aromatic heterocyclic motif found in many natural and synthetic molecules with various pharmacological activities including antimicrobial, anticancer, anti-inflammatory, antiviral, antioxidant activities, etc.16-20 Ellipticine, as a typical example, is a natural carbazole alkaloid exhibiting powerful antiproliferative properties and has been used clinically as an anticancer drug. A number of ellipticine derivatives have also been designed and synthesized with improved anticancer activities against a panel of cancer cell lines.21-23 Thus it is speculated that the fusion of a carbazole moiety with the molecule of UA would probably change its physicochemical properties and lead to better biological activities. On the other hand, previous studies indicated that the incorporation of polar moiety onto the C-3 or C-28 position might improve the water solubility and thus clinical utility.24,25 In a former literature, UA derivatives were conjugated with different amino acids on the C-28 carboxyl groups. Some
derivatives with amino groups (positively charged) exhibited more potent cytotoxic activity than UA and the derivatives bearing carboxyl groups (negatively charged).26 Therefore, the introduction of amine moieties onto C-28 carboxyl groups of UA derivatives might also be benefical to their biological activities. Prompted by the above facts and in continuation with our efforts towards potential natural product-derived antimicrobial and anticancer agents, a series of novel carbazole derivatives of UA were designed and synthesized. The reaction sequence employed in the synthesis of the target compounds was shown in Scheme 1. First, the starting material UA (1) was oxidized by Jone’s reagent to afford 3-oxo-ursolic acid (2) in 76% yield. Subsequently, a series of carbazole derivatives of UA (3a-h) were synthesized from compound 2 and different substituted phenylhydrazine hydrochlorides in 55-65% yields through Fisher indole reactions. Further the esterification of the carboxyl groups of compounds 3a-h proceeded under known procedure to afford the corresponding ester products 4a-h in 82-89% yields. On the other hand, compounds 3a-h were converted to the corresponding amide derivatives 5a-h in 73-85% yields by reacting with 3-(dimethylamino)-1-propylamine (DMAPA) in the presence of 1-hydroxybenzotriazole (HOBt) and dicyclohexylcarbodiimide (DCC). All the synthesized target compounds were purified by recrystallization or silica gel column chromatography, and the structures of these compounds were characterized by their HR-MS, IR and 1H NMR spectra. Next, all the synthesized compounds were screened for the antimicrobial activity. The microorganisms used in the study were two Gram-positive bacteria (Bacillus subtilis CGMCC 1.1162, Staphylococcus aureus CGMCC 1.1361), two Gram-negative bacteria (Escherichia coli CGMCC 1.1571, Pseudomonas fluorescens CGMCC 1.1828) and three fungi (Candida albicans
CGMCC 2.2086, Candida tropicalis CGMCC 2.3967 and Aspergillus niger CGMCC 3.316). The minimum inhibitory concentrations (MICs) of the target compounds were determined through a modified broth microdilution method.27 The widely used antibacterial drug amikacin sulfate and antimycotic drug ketoconazole were co-assayed as positive controls against the tested bacteria and fungi, respectively. The results of the antimicrobial assay were shown in Table 1. As illustrated in Table 1, most of the synthesized compounds exhibited inhibitory activity against both Gram-positive and Gram-negative bacteria with MIC values < 100 µg/ml. Among them, compounds 3a, 3b, 4a, 4b and 5a-f displayed considerable antibacterial activity against at least one tested bacteria with MIC values ranging from 3.9 to 15.6 µg/ml. Specially, compound 5b exerted comparatively the highest inhibitory effect against B. subtilis with MIC value of 3.9 µg/ml, near to that of positive control. The compound also showed significant inhibition (MIC: 7.8 µg/ml) against the other three bacterial strains. In addition, compounds 5a and 5c also registered same inhibitory potency (MIC: 7.8 µg/ml) against B. subtilis and S. aureus, respectively. On the other hand, compounds 3c-h, 4c-h, 5g and 5h were found to display moderate, weak or even no inhibition against the four bacterial strains. Concerning the antifungal activity, most of the synthesized compounds showed mild or no inhibition against the three tested fungi. Among them, compounds 3d, 4e, 5a, 5b, 5c and 5f exhibited moderate inhibitory activity (MIC: 31.2 µg/ml) against A. niger. In addition, 5a and 5b were also inhibitory to C. albicans and/or C. tropicalis at the same concentration. From the in vitro antimicrobial activity data, preliminary structure-activity relationship of the synthesized compounds was studied. A number of derivatives showed enhanced activities than the parent intermediate 2, which indicated that the incorporation of carbazole moiety to the molecule
was beneficial to the antimicrobial activity. The results also suggested that the substituents at C-28 position of these derivatives had an important effect on their antimicrobial activity. In general, the derivatives with N-(dimethylamino)propyl amide side chains (5a-h) exhibited higher antibacterial activity than other derivatives with carboxyl groups (3a-h) and methyl ester groups (4a-h). For example, compound 5b exhibited significant activity against four tested bacteria (MIC: 3.9 ~ 7.8 µg/ml), while its analogs 3b and 4b were only moderately inhibitory to the four strains (MIC: 15.6 ~ 31.2 µg/ml). Moreover, the antimicrobial activity of these UA derivatives were also substantially influenced by the substituents on the phenyl rings. Among derivatives 5a-h, compounds 5b and 5c with electron-withdrawing substituents (F and Cl) were more potent against tested bacteria than those with electron-donating substituents (CH3, OCH3 and OC2H5). However, compound 5d with relatively weak electron-withdrawing substituent (Br) only exhibited moderate antimicrobial activity. Similar results could be observed in derivatives 3a-h and 4a-h. The results indicated that the appropriate electron density on the phenyl ring of these derivatives might be closely related to high antimicrobial activity. The target compounds were also evaluated for their in vitro cytotoxicity against two human hepatocarcinoma cell lines (SMMC-7721 and HepG2) using MTT assay method.28 The proverbial anticancer drug doxorubicin was used as the positive control. The results of the test compounds expressed as IC50 values (concentration required to inhibit tumor cell proliferation by 50%) are presented in Table 2. From the results, these UA derivatives exhibited varying degrees of inhibitory activity against the two cell lines. Compounds 5a-e and 5h showed significant cytotoxic activity with IC50 values below 10 µM. Notably, compound 5e was the most potent compound in the series with IC50 values of 1.08 ± 0.22 and 1.26 ± 0.17 µM against SMMC-7721 and HepG2
cells, respectively, which were comparable to those of positive control doxorubicin. In addition, compound 5e was also assayed for the in vitro cytotoxic activity against the noncancerous human hepatocyte cell line LO2, which showed reduced cytotoxicity with IC50 value of 5.75 ± 0.48 µM. Compounds 3a-f, 3h and 5g also showed moderate cytotoxicity against one or two cancer cell lines. However, nearly all of compounds 4a-h were inactive to tested cancer cells with IC50 values > 100 µM. The aforementioned results suggested that the cytotoxic activity of these derivatives was also markedly influenced by their structural properties. Similar to the antimicrobial activity, the cytotoxic activity of amide derivatives 5a-h was found to be much more potent than the other two series of derivatives, indicating that the N-(dimethylamino)propyl moiety was crucial for their cytotoxic activity. Contrarily, the esterification of the C-28 carboxyl group would substantially decrease the cytotoxicity of the derivatives. A possible explanation might be the electrostatic interaction between the derivatives and the cancer cells. The outer surface of assayed cancer cells presented net negative charge in physiological environment, which facilitated the accumulation of positively charged compounds around cells.26,29,30 Therefore, compounds 5a-h with amine moiety (positively charged) would obtain higher local drug concentration and enhanced cytotoxic activity. On the other hand, the substituents on the benzene rings of these derivatives affected the cytotoxic activity to a certain extent. For compounds 5a-h, derivatives with relatively more lipophilic substituents such as H atom (5a), halogen atoms (5b-d), CH3 (5e) and OC2H5 (5h) exhibited stronger inhibitory activity than those with more polar methoxy groups (5f and 5g). The derivatives 3a-h displayed similar results as well. The introduction of substituents with higher lipophilicity on the indole benzene ring would probably lead to better membrane permeability, and
thus increased their pharmaceutical effects.31,32 As for methoxy groups, the relocation of the methoxy group from the p-position (3f and 5f) to the o-position (3g and 5g) decreased the cytotoxic activity remarkably, which suggested that p-substituted methoxy substituent was more favorable to the cytotoxicity than o-substituted methoxy group. In summary, a series of novel carbazole derivatives of ursolic acid were designed, synthesized, and evaluated for their in vitro antimicrobial and cytotoxic activities. As a result, a number of derivatives exhibited promising antimicrobial and/or cytotoxic activities when compared with the positive
controls.
Among
them,
compound
5b
with
5-fluoroindole
moiety
and
N-(dimethylamino)propyl amide side chain showed pronounced antibacterial activity against the tested bacteria. Moreover, its analog 5e with 5-methylindole moiety displayed potent cytotoxic activity against two human hepatocarcinoma cell lines comparable to doxorubicin. The activities of these derivatives were closely relevant to their structural properties. These findings would open a new avenue for the development of this class of UA derivatives as prospective antimicrobial or anticancer agents. Further investigations, including extensive structural modifications and in-depth SAR analysis, cytotoxic assay against other tumor cell lines and pharmacological studies are to be performed for discovering new derivatives with better activities and revealing their exact mechanism of action.
Acknowledgements The authors are grateful to the National Natural Science Foundation of China for financial support (Project: 31000273). This work was also supported by a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). The authors
would like to thank the Advanced Analysis & Testing Center of Nanjing Forestry University for the measurements of NMR data.
Supplementary data Supplementary data associated with this article can be found, in the online version, at http://
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Table 1 Antimicrobial activity of synthesized compounds (MIC values are in µg/ml) Compd No.
Antibacterial activity
Antifungal activity
Gram positive bacteria
Gram negative bacteria
B. subtilis
S. aureus
E. coli
P. fluorescens
A. niger
C. albicans
C. tropicalis
2
31.2
62.5
31.2
62.5
>100
>100
>100
3a
15.6
31.2
15.6
15.6
>100
>100
>100
3b
15.6
15.6
15.6
31.2
62.5
62.5
>100
3c
31.2
31.2
31.2
31.2
>100
>100
>100
3d
31.2
62.5
62.5
62.5
31.2
62.5
62.5
3e
62.5
62.5
31.2
62.5
>100
>100
>100
3f
31.2
31.2
31.2
62.5
>100
>100
62.5
3g
62.5
62.5
62.5
62.5
>100
>100
>100
3h
62.5
62.5
62.5
62.5
>100
>100
>100
4a
31.2
15.6
31.2
62.5
62.5
62.5
62.5
4b
31.2
15.6
31.2
31.2
62.5
62.5
62.5
4c
31.2
62.5
>100
62.5
62.5
>100
>100
4d
62.5
31.2
>100
62.5
>100
>100
>100
4e
31.2
31.2
>100
62.5
31.2
>100
62.5
4f
31.2
31.2
62.5
>100
>100
>100
>100
4g
31.2
31.2
>100
>100
>100
>100
>100
4h
31.2
31.2
>100
62.5
>100
>100
>100
5a
7.8
15.6
15.6
31.2
31.2
31.2
62.5
5b
3.9
7.8
7.8
7.8
31.2
31.2
31.2
5c
15.6
7.8
15.6
31.2
31.2
62.5
62.5
5d
31.2
31.2
31.2
15.6
62.5
>100
62.5
5e
15.6
31.2
31.2
31.2
62.5
>100
>100
5f
15.6
31.2
31.2
31.2
31.2
62.5
62.5
5g
31.2
62.5
31.2
31.2
>100
>100
>100
5h
31.2
62.5
31.2
62.5
>100
>100
>100
Amikacin
0.9
0.9
1.9
0.9
−
−
−
Ketoconazole
−
−
−
−
7.8
3.9
3.9
Table 2 IC50 values of the synthesized compounds against two hepatocarcinoma cell lines. Compound
IC50 value (µM) SMMC-7721
HepG2
2
>100
>100
3a
40.36 ± 1.72
25.77 ± 0.83
3b
16.31 ± 0.52
15.21 ± 0.90
3c
29.88 ± 1.43
30.95 ± 2.78
3d
62.63 ± 2.89
38.40 ± 1.86
3e
13.80 ± 0.85
17.03 ± 2.27
3f
31.73 ± 0.98
34.60 ± 0.77
3g
>100
>100
3h
24.48 ± 1.58
33.05 ± 2.24
4a
>100
>100
4b
>100
97.00 ± 2.02
4c
>100
>100
4d
>100
>100
4e
>100
>100
4f
>100
>100
4g
>100
>100
4h
>100
>100
5a
2.12 ± 0.35
2.78 ± 0.21
5b
4.60 ± 1.17
6.03 ± 1.12
5c
2.78 ± 0.31
4.02 ± 0.86
5d
6.80 ± 1.09
5.65 ± 1.43
5e
1.08 ± 0.22
1.26 ± 0.17
5f
12.77 ± 0.53
11.37 ± 0.69
5g
36.18 ± 2.01
88.78 ± 3.98
5h
4.88 ± 0.72
6.25 ± 1.37
Doxorubicin
0.62 ± 0.16
0.77 ± 0.12
Legend Scheme 1. Synthetic route for the carbazole derivatives (3a-h, 4a-h and 5a-h) from ursolic acid. Reagents and conditions: (a) Jone’s reagent, acetone, 0 °C, 5 h; (b) substituted phenylhydrazine hydrochloride, EtOH, conc. HCl, reflux for 3h; (c) i. SOCl2, benzene, reflux for 3h; ii. Methanol, reflux for 2h; (d) DMAPA, HOBt, DCC, CH2Cl2, rt, 12h.
Scheme 1.
Graphic Abstract
