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Semisynthesis of salviandulin E analogues and their antitrypanosomal activity
Bioorganic & Medicinal Chemistry Letters 24 (2014) 442–446
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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Semisynthesis of salviandulin E analogues and their antitrypanosomal activity Yutaka Aoyagi a,⇑, Koji Fujiwara b, Akira Yamazaki b, Naoko Sugawara b, Reiko Yano a, Haruhiko Fukaya b, Yukio Hitotsuyanagi b, Koichi Takeya b,⇑, Aki Ishiyama c, Masato Iwatsuki c, Kazuhiko Otoguro c, ¯ mura d Haruki Yamada d, Satoshi O a
College of Pharmacy, Kinjo Gakuin University, 2-1723 Omori, Moriyama-ku, Nagoya, Aichi 463-8521, Japan School of Pharmacy, Tokyo University of Pharmacy & Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan Research Center for Tropical Diseases, Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan d Kitasato Institute for Life Sciences, Graduate School of Infection Control Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan b c
a r t i c l e
i n f o
Article history: Received 30 October 2013 Revised 9 December 2013 Accepted 13 December 2013 Available online 21 December 2013 Keywords: Semisynthesis Antitrypanosomal activity Neoclerodane diterpene Salvia leucantha Salviandulin E
a b s t r a c t A series of analogues of salviandulin E, a rearranged neoclerodane diterpene originally isolated from Salvia leucantha (Lamiaceae), were prepared and their in vitro activity against Trypanosoma brucei brucei was evaluated with currently used therapeutic drugs as positive controls. One of the 19 compounds prepared and assayed in the present study, butanoyl 3,4-dihydrosalviandulin E analogue was found to be a possible candidate for an antitrypanosomal drug with fairly strong antitrypanosomal activity and lower cytotoxicity. Ó 2013 Elsevier Ltd. All rights reserved.
Human African trypanosomiasis (HAT), also known as sleeping sickness, caused by infection of protozoan parasites Trypanosoma brucei rhodesiense or T. b. gambiense is a major threat to the communities in the sub-Saharan area of Africa.1,2 After infection, the parasites invade the central nerve system, causing behavioral changes, coma (sleeping sickness), and eventually death, if left untreated. All the currently used drugs to treat HAT are not satisfactory with their poor efficacy and undesirable side effects.3 Thus, there is an urgent need for better drugs for the more effective treatment of HAT, which are safe, cheap, and easy to administer. Previously, we reported that some natural products showed potent antitrypanosomal properties.4 Among such natural products is salviandulin E (1), a rearranged neoclerodane diterpene having a unique carbon skeleton from Salvia leucantha (Lamiaceae), showed a potent antitrypanosomal activity (IC50 0.72 lg/mL) against T. b. brucei GUTat 3.1 parasites. The plant is rich in neoclerodane diterpenes.5,6 Another similar diterpene isolated from the plant, 6,7-dehydrodugesin A (2),7 having no oxygen atom at 2, however, showed no antitrypanosomal activity (IC50 >12.5 lg/mL). This fact suggested a plausible role of the 2oxygen functionality in the antitrypanosomal activity. Therefore, in the present study, to examine the effect of the roles of 2-oxygen ⇑ Corresponding authors. E-mail addresses: [email protected] (Y. Aoyagi), [email protected] (K. Takeya). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.12.052
function on the activity, we prepared a series of analogues from 2oxygenated diterpene 1 and its hydrogenated analogue 11, and evaluated their in vitro activities against T. b. brucei GUTat 3.1 with currently commonly used therapeutic drugs as positive controls. 15
O 16
14 20 13 1
R 2
10
4
O 17
5
3
12
11 9
19 6
8
O
7
O 18O salviandulin E (1)
R = OH
6,7-dehydrodugesin A (2 )
R=H
The absolute configuration of 1 was determined by the X-ray crystallography of the salviandulin p-bromobenzoyl ester (3), prepared as shown in Scheme 1, and was established to be as shown in Figure 1.8 A series of analogues of 1 were prepared as shown in Scheme 2. Thus, conventional acylation of 1 gave 4–6, reactions with alkyl
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Y. Aoyagi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 442–446
O
O Br O
HO
O O
O
O
p-bromobenzoyl chloride O
CH 2Cl2:pyridine (3:1)
O O
O
O
3
salviandulin E (1) Scheme 1. p-Bromobenzoylation of salviandulin E (1).
Figure 1. Ortep representation for 3.
O
O
R
O
O O
R (RCO) 2O pyridine
O
O
MOMCl Et3 N
O
O
O
O
1
NaBH 4 MeOH
O
O
HO
O O
O
products yields (%) R = Et 7 20 63 Bu 8 Ph 9 quant. O
O
O
O
R-N=C=O
HO
O
O
O O
O
O O products yields (%) R = Me 4 96 Et 5 73 n Pr 6 82
O
H N
10 56% Scheme 2. Preparation of salviandulin E analogues.
O H O
O
11 80%
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Figure 2. Ortep representation for 11.
and phenyl isocyanides gave the corresponding urethanes (7–9), and methoxymethylation gave 10. For the evaluation of the effect of conformation of A/c-lactone ring system on the antitrypanosomal activity, hydrogenation of 1 was carried out to give 3,4-dihydrosalviandulin E (11) as shown in Fig. 2.9 The crystallographic analysis of 11 revealed that the hydrogen was added from a-face as shown in Scheme 2. The relationship between the 3D-structures of 3 and 11 as revealed by the X-ray analysis was studied by aligning the common B/C ring system by using Sybyl 7.3 software.10 As shown in Fig. 3, the conformation of 3 deviates from that of 11. Analogues of 11 were prepared according to Scheme 3. Thus, acylation of 11 gave 12–14 and the reaction with isocyanides gave 15–17. When 11 was treated with DMSO-acyl anhydrides at room temperature, the products were the enol esters 19–21. When the reaction was performed at 78 °C, 1,2-dehydrated analogue (22) was obtained along with 19. The methoxymethylation of 11 gave 18. The in vitro antitrypanosomal activities of natural products, salviandulin E (1) and 2-desoxysalviandulin E (2, 6,7-dehydrodugeshin A), and its 19 analogues prepared in the present study were assayed by using GUTat3.1 (T. b. brucei),11 along with the currently used therapeutic drugs, pentamidine, suramin and eflornithine, as the positive controls. The average values of the IC50 lg/mL of antitrypanosomal activity on GUTat3.1 and of
cytotoxicity on MRC-5 are presented in Table 1, along with the selectivity index (cytotoxicity IC50/antitrypanosomal activity IC50). 2-Desoxysalviandulin E (2) and 22 showed no activity, suggesting that the oxygen functionality at 2-position was essential for this series of compounds to show antitrypanosomal activity. The antitrypanosomal activities and cytotoxicities of the salviandulin E analogues 4–7, and 10 were about the same as those of the mother compound, salviandulin E (1). The small differences in the activities noted among them might be attributed to the slight deviation of the location of the 2-oxygen function caused by the introduction of different groups, resulting in slight changes in the activity. Similar results were observed among the activities of 19–21. The fact implies that the double bond in the 2-oxygen-bearing ring fixes the location of the 2-oxygen group, which is only slightly affected by the group introduced to the 2-oxygen to cause slight changes in the activity. This fact also supports our hypothesis that the 2-oxygen or its location in the molecule plays an important role in deciding the antitrypanosomal activity of this series of compounds. No antitrypanosomal activity was observed in 3,4-dihydrosalviandulin E (11). Among its derivatives (12–18), the changes in the activity caused by the different groups introduced are more conspicuous. In those compounds, due to the changes in the conformation of A/c-lactone ring system or the absence of the double bond in the 2-O bearing ring, the location of 2oxygen group may now be more easily affected by the groups introduced to modify the activity of the compound. Thus 15 and 18 showed no activity, whereas butanoyl 3,4-dihydrosalviandulin E analogue (14) showed a potent antitrypanosomal activity with the IC50 of 0.055 lg/mL (N = 4, SD ± 0.025), with the selectivity index of 1236 (Table 1). Of the three currently used therapeutic drugs used as the control in the present assay, suramin and eflornithine showed rather weak antitrypanosomal activity, implying that the antitrypanosomal activity is not their major therapeutic effect expected: they may cure the disease by some other way. Our present results give positive support to our hypothesis that the antitrypanosomal activity of this series of compounds and the location and the length of alkyl chain of the 2-oxygen function may be closely related. We also consider that 14 can be a possible candidate for an antitrypanosomal drug. Further studies including in vivo tests of the compound 14 in animal models are now in progress. Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (To Y. A.) and funds from the Drugs for Neglected Diseases initiative (DNDi). Our thanks are due to Ms Miyuki Namatame (Kitasato University) for technical assistance.
Figure 3. Aligned stereodrawing of 3 and 11, which were produced by X-ray crystallographic analysis.
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Y. Aoyagi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 442–446
O
O
R
O
O O
H N
(RCO) 2O pyridine
MOMCl Et3 N
O
O
O
H O
O
O H O O 11 DMSO -78 °C (MeCO) 2O
O
products yields (%) R = Et 15 85 n Pr 16 63 Ph 17 70
O
O H O
O
R-N=C=O
HO
O
O
O O
O
H O O products yields (%) R = Me 12 66 Et 13 65 n Pr 14 59
O
R
DMSO (RCO)2 O
O
rt R
O
O O
O
O H O
18 62% O
+
19 24%
O H O
O
22 26%
O
products yields (%) R = Me 19 41 Et 20 26 nPr 21 12
Scheme 3. Preparation of dehydrosalviandulin E (11) analogues.
Table 1 IC50 values of in vitro antitrypanosomal activities (Trypanosoma brucei brucei GUTat 3.1) and of cytotoxicities (MRC-5 cells) of salviandulin E (1), its analogues (2 and 4–22), and currently used drugs, and their ratios. Compounds
Salviandulin E (1) 2 4 5 6 7 8 9 10 11 12 13 14c 15 16 17 18 19 20 21 22 Pentamidine suramina Eflornithinea a b c
IC50 (lg/mL)
Selectivity index (SI)
Antitrypanosomal activity (GUTat 3.1)
Cytotoxicity (MRC-5)
Cytotoxicity IC50/antitrypanosomal activity IC50
0.72 >12.5 0.69 2.39 1.04 0.20 6.08 7.17 1.15 >12.5 7.73 5.33 0.06 >12.5 7.77 10.55 >12.5 4.29 5.56 3.89 >12.5 0.0017 1.58 2.27
0.84 16.17 0.84 1.41 1.05 0.18 44.29 17.27 2.00 NDb >100 63.48 68.00 NDb 66.16 >100 NDb 17.78 22.59 21.56 NDb 5.7 >100 >100
1.17 — 1.22 0.59 1.01 0.90 7.29 2.41 1.73 — >12.9 11.91 1236 — 8.51 >9.5 — 4.14 4.06 5.54 — 3353 >63 >44
Ref. 4a. Not determined. Experimental data for 14: IC50 was 0.055 lg/mL (N = 4, SD ± 0.025).
References and Notes 1. http://www.dndi.org/ 2. Steverding, D. Steverding Parasites & Vectors 2010, 3, 15. and the references cited therein. 3. Fairamb, A. H. Trends Parasitol. 2003, 19, 488. 4. (a) Otoguro, K.; Ishiyama, A.; Namatame, M.; Nishihara, A.; Furusawa, T.; Masuma, R.; Shiomi, K.; Takahashi, Y.; Yamada, H.; Omura, S. J. Antibiot. 2008,
61, 372; (b) Ishiyama, A.; Otoguro, K.; Namatame, M.; Nishihara, A.; Furusawa, T.; Masuma, R.; Shiomi, K.; Takahashi, Y.; Ichimura, M.; Yamada, H.; Omura, S. J. Antibiot. 2008, 61, 627; (c) Otoguro, K.; Iwatsuki, M.; Ishiyama, A.; Namatame, M.; Nishihara-Tukashima, A.; Kiyohara, H.; Hashimoto, T.; Asakawa, Y.; Omura, S.; Yamada, H. Phytochemistry 2011, 72, 2024; (d) Otoguro, K.; Ishiyama, A.; Iwatsuki, M.; Namatame, M.; Nishihara-Tukashima, A.; Kiyohara, H.; Hashimoto, T.; Asakawa, Y.; Omura, S.; Yamada, H. J. Nat. Med. 2012, 66, 377; (e) Otoguro, K.; Iwatsuki, M.; Ishiyama, A.; Namatame, M.; Nishihara-
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5.
6.
7.
8.
9.
Y. Aoyagi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 442–446 Tukashima, A.; Kiyohara, H.; Hashimoto, T.; Asakawa, Y.; Omura, S.; Yamada, H. J. Nat. Med. 2012, 66, 558. and the references cited therein. (a) Esquivel, B.; Dominguez, M.; Hernandez-Ortega, S.; Toscano, R. A.; Rodriguez-Hahn, L. Tetrahedron 1994, 50, 111593; (b) Narukawa, Y.; Hatano, K.; Takeda, T. J. Nat. Med. 2006, 60, 206. (a) Aoyagi, Y.; Yamazaki, A.; Nakatsugawa, C.; Fukaya, H.; Takeya, K.; Kawauchi, S.; Izumi, H. Org. Lett. 2008, 10, 4429; (b) Aoyagi, Y.; Yamazaki, A.; Kato, R.; Tobe, F.; Fukaya, H.; Nishikawa, T.; Nakahashi, A.; Miura, N.; Monde, K.; Takeya, K. Tetrahedron Lett. 2011, 52, 1851. Xu, G.; Peng, L.; Niu, X.; Zhao, Q.; Li, R.; Sun, H. Helv. Chim. Acta 2004, 87, 949. In the present study, 0.018 g of 6,7-dehydrodugesin A (2) was obtained from the aerial parts of this plant (dry weight 18.7 kg). The physical and spectral data of 2 were identical with those reported. To a solution of salviandulin 1 (0.02 g, 0.057 mmol) in CH2Cl2-pyridine (3:1, 2 mL) were added p-bromobenzoyl chloride (0.066 g, 0.30 mmol) and DMAP (0.008 g, 0.0065 mmol) sequentially. The reaction mixture was then stirred at rt for 4 h. Usual work-up and subsequent silica gel column chromatography (CHCl3:MeOH = 10:1) gave the corresponding p-bromobenzoate (3) in 92% yield. Colorless amorphous solid, [a]D: +37.9°(c 0.21, CHCl3), IR(film) m max 1759 (C@O), 1721 (C@O) cm1. HRESIMS: m/z 535.0391 [(M+H)+, calcd for C27H20O7Br, 535.0392]. 1H NMR (pyridine-d5, 500 MHz): d 2.05(3H, s), 2.68(1H, br dd), 3.32(1H, dd, 15.4, 5.3), 3.42(1H, d, 8.4), 4.10(1H, d, 8.4), 5.51(1H, d, 9.1), 5.68(1H, ddd, 10.2, 5.3, 2.0), 6.53(1H, s), 6.72(1H, s), 7.03(1H, d, 9.1), 7.63(1H, s), 7.68(1H, s), 7.74(2H, d, 8.4), 7.99(1H, s), 8.08(2H, d, 8.4). 13C NMR (pyridined5, 125 MHz): d 16.7 (q), 34.2 (t), 47.0 (s), 70.7 (d), 75.6 (t), 76.4 (d), 109.2 (d), 121.5 (s), 122.1 (d), 126.3 (s), 127.7 (s), 128.9 (s), 129.0 (s), 129.0 (s), 129.5 (d), 131.8 (d), 132.3 (d), 133.6 (s), 138.8 (d), 142.5 (d), 145.1 (d), 159.4 (s), 165.1 (s), 168.0 (s), 171.5 (s). Crystallographic data for 3 has been deposited with the Cambridge Crystallographic Data Centre as supplementary publication No. CCDC 966202. Copies of the data can be obtained, free of charge, on application to the Director, CCDC (e-mail: [email protected]). To a MeOH solution (5 mL) of 1 (0.05 g, 0.14 mmol) was added sodium borohydride (0.006 g, 0.16 mmol) at 0°C and the mixture was stirred at the
same temperature for 6 h. Then, H2O (10 mL) was added to the mixture at rt Usual work-up followed by silica gel column chromatography (hexanes: Me2CO = 1.7:1) of the resulting product gave 11 in 80% yield. Colorless needles, mp 119–121 °C, ½a25 D : 125.2 (c 0.11, CHCl3), IR (film): m max 1754 (C@O) cm1, HRESIMS: m/z 355.1163 [(M+H)+, calcd for C20H19O7, 355.1182]. 1H NMR (CDCl3, 500 MHz): d 2.05 (3H, s), 2.24 (1H, ddd, 14.2, 6.6, 5.8), 2.32 (1H, ddd, 14.2, 6.4, 3.7), 2.58 (1H, dd, 17.5, 3.8), 2.71 (1H, dd, 17.5, 6.9), 3.00 (1H, dd, 5.4, 1.6), 3.55 (2H, s), 4.07 (1H, m), 5.56 (1H, d, 9.3), 6.21 (1H, d, 1.0), 6.29 (1H, s), 6.80 (1H, dd, 9.3, 1.5), 7.43 (1H, dd, 1.7, 1.7), 7.52 (1H, d, 0.7). 13C NMR (CDCl3, 125 MHz): d 16.9 (q), 29.6 (t), 36.6 (t), 44.9 (d), 46.2 (s), 65.7 (d), 75.1 (t), 76.3 (d), 108.2 (d), 120.1 (s), 121.4 (d), 125.2 (s), 125.3 (s), 128.9 (d), 136.8 (s), 140.7 (d), 144.6 (d), 158.8 (s), 171.3 (s), 177.7 (s). Crystallographic data for 11 has been deposited with the Cambridge Crystallographic Data Centre as supplementary publication No. CCDC 966201. Copies of the data can be obtained, free of charge, on application to the Director, CCDC (e-mail: [email protected]). 10. Molecular modeling software SYBYL7.3 (Tripos associates, St. Louis, MI, USA) was employed. The X-ray structures obtained were aligned so that A and clactone rings were superimposed to form a common structure by using the database aligned method. 11. In vitro antitrypanosomal activities against T. b. brucei strain GUTat 3.1 were measured as described previously.4 In brief, the strain was cultured in Iscove’s modified Dulbecco’s medium (IMDM) with various supplements and 10% heatinactivated fetal bovine serum at 37 °C, under 5.0% CO2-95% air. Subsequently, 95 ll of the trypanosomes suspension (2.0–2.5 104 trypanosomes/ml) was transferred in a 96-well microtiter plate and 5.0 ll of a test compound solution (dissolved in 5.0% dimethylsulfoxide) was added and incubated for 72 h at 37 °C. Then, 10 ll of the fluorescent dye AlamarBlue was added to each well. After incubation for 3–6 h, the resulting solution was read at 528/20 nm excitation wavelengths and 590/30 nm emission wavelengths by an FLx800 fluorescence microplate reader (Bio-Tek Instruments, Inc. Winooski, VT, USA).The IC50 values were determined using the fluorescent plate reader software (KC-4, Bio-Tek).
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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Semisynthesis of salviandulin E analogues and their antitrypanosomal activity Yutaka Aoyagi a,⇑, Koji Fujiwara b, Akira Yamazaki b, Naoko Sugawara b, Reiko Yano a, Haruhiko Fukaya b, Yukio Hitotsuyanagi b, Koichi Takeya b,⇑, Aki Ishiyama c, Masato Iwatsuki c, Kazuhiko Otoguro c, ¯ mura d Haruki Yamada d, Satoshi O a
College of Pharmacy, Kinjo Gakuin University, 2-1723 Omori, Moriyama-ku, Nagoya, Aichi 463-8521, Japan School of Pharmacy, Tokyo University of Pharmacy & Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan Research Center for Tropical Diseases, Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan d Kitasato Institute for Life Sciences, Graduate School of Infection Control Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan b c
a r t i c l e
i n f o
Article history: Received 30 October 2013 Revised 9 December 2013 Accepted 13 December 2013 Available online 21 December 2013 Keywords: Semisynthesis Antitrypanosomal activity Neoclerodane diterpene Salvia leucantha Salviandulin E
a b s t r a c t A series of analogues of salviandulin E, a rearranged neoclerodane diterpene originally isolated from Salvia leucantha (Lamiaceae), were prepared and their in vitro activity against Trypanosoma brucei brucei was evaluated with currently used therapeutic drugs as positive controls. One of the 19 compounds prepared and assayed in the present study, butanoyl 3,4-dihydrosalviandulin E analogue was found to be a possible candidate for an antitrypanosomal drug with fairly strong antitrypanosomal activity and lower cytotoxicity. Ó 2013 Elsevier Ltd. All rights reserved.
Human African trypanosomiasis (HAT), also known as sleeping sickness, caused by infection of protozoan parasites Trypanosoma brucei rhodesiense or T. b. gambiense is a major threat to the communities in the sub-Saharan area of Africa.1,2 After infection, the parasites invade the central nerve system, causing behavioral changes, coma (sleeping sickness), and eventually death, if left untreated. All the currently used drugs to treat HAT are not satisfactory with their poor efficacy and undesirable side effects.3 Thus, there is an urgent need for better drugs for the more effective treatment of HAT, which are safe, cheap, and easy to administer. Previously, we reported that some natural products showed potent antitrypanosomal properties.4 Among such natural products is salviandulin E (1), a rearranged neoclerodane diterpene having a unique carbon skeleton from Salvia leucantha (Lamiaceae), showed a potent antitrypanosomal activity (IC50 0.72 lg/mL) against T. b. brucei GUTat 3.1 parasites. The plant is rich in neoclerodane diterpenes.5,6 Another similar diterpene isolated from the plant, 6,7-dehydrodugesin A (2),7 having no oxygen atom at 2, however, showed no antitrypanosomal activity (IC50 >12.5 lg/mL). This fact suggested a plausible role of the 2oxygen functionality in the antitrypanosomal activity. Therefore, in the present study, to examine the effect of the roles of 2-oxygen ⇑ Corresponding authors. E-mail addresses: [email protected] (Y. Aoyagi), [email protected] (K. Takeya). 0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.12.052
function on the activity, we prepared a series of analogues from 2oxygenated diterpene 1 and its hydrogenated analogue 11, and evaluated their in vitro activities against T. b. brucei GUTat 3.1 with currently commonly used therapeutic drugs as positive controls. 15
O 16
14 20 13 1
R 2
10
4
O 17
5
3
12
11 9
19 6
8
O
7
O 18O salviandulin E (1)
R = OH
6,7-dehydrodugesin A (2 )
R=H
The absolute configuration of 1 was determined by the X-ray crystallography of the salviandulin p-bromobenzoyl ester (3), prepared as shown in Scheme 1, and was established to be as shown in Figure 1.8 A series of analogues of 1 were prepared as shown in Scheme 2. Thus, conventional acylation of 1 gave 4–6, reactions with alkyl
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Y. Aoyagi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 442–446
O
O Br O
HO
O O
O
O
p-bromobenzoyl chloride O
CH 2Cl2:pyridine (3:1)
O O
O
O
3
salviandulin E (1) Scheme 1. p-Bromobenzoylation of salviandulin E (1).
Figure 1. Ortep representation for 3.
O
O
R
O
O O
R (RCO) 2O pyridine
O
O
MOMCl Et3 N
O
O
O
O
1
NaBH 4 MeOH
O
O
HO
O O
O
products yields (%) R = Et 7 20 63 Bu 8 Ph 9 quant. O
O
O
O
R-N=C=O
HO
O
O
O O
O
O O products yields (%) R = Me 4 96 Et 5 73 n Pr 6 82
O
H N
10 56% Scheme 2. Preparation of salviandulin E analogues.
O H O
O
11 80%
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Y. Aoyagi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 442–446
Figure 2. Ortep representation for 11.
and phenyl isocyanides gave the corresponding urethanes (7–9), and methoxymethylation gave 10. For the evaluation of the effect of conformation of A/c-lactone ring system on the antitrypanosomal activity, hydrogenation of 1 was carried out to give 3,4-dihydrosalviandulin E (11) as shown in Fig. 2.9 The crystallographic analysis of 11 revealed that the hydrogen was added from a-face as shown in Scheme 2. The relationship between the 3D-structures of 3 and 11 as revealed by the X-ray analysis was studied by aligning the common B/C ring system by using Sybyl 7.3 software.10 As shown in Fig. 3, the conformation of 3 deviates from that of 11. Analogues of 11 were prepared according to Scheme 3. Thus, acylation of 11 gave 12–14 and the reaction with isocyanides gave 15–17. When 11 was treated with DMSO-acyl anhydrides at room temperature, the products were the enol esters 19–21. When the reaction was performed at 78 °C, 1,2-dehydrated analogue (22) was obtained along with 19. The methoxymethylation of 11 gave 18. The in vitro antitrypanosomal activities of natural products, salviandulin E (1) and 2-desoxysalviandulin E (2, 6,7-dehydrodugeshin A), and its 19 analogues prepared in the present study were assayed by using GUTat3.1 (T. b. brucei),11 along with the currently used therapeutic drugs, pentamidine, suramin and eflornithine, as the positive controls. The average values of the IC50 lg/mL of antitrypanosomal activity on GUTat3.1 and of
cytotoxicity on MRC-5 are presented in Table 1, along with the selectivity index (cytotoxicity IC50/antitrypanosomal activity IC50). 2-Desoxysalviandulin E (2) and 22 showed no activity, suggesting that the oxygen functionality at 2-position was essential for this series of compounds to show antitrypanosomal activity. The antitrypanosomal activities and cytotoxicities of the salviandulin E analogues 4–7, and 10 were about the same as those of the mother compound, salviandulin E (1). The small differences in the activities noted among them might be attributed to the slight deviation of the location of the 2-oxygen function caused by the introduction of different groups, resulting in slight changes in the activity. Similar results were observed among the activities of 19–21. The fact implies that the double bond in the 2-oxygen-bearing ring fixes the location of the 2-oxygen group, which is only slightly affected by the group introduced to the 2-oxygen to cause slight changes in the activity. This fact also supports our hypothesis that the 2-oxygen or its location in the molecule plays an important role in deciding the antitrypanosomal activity of this series of compounds. No antitrypanosomal activity was observed in 3,4-dihydrosalviandulin E (11). Among its derivatives (12–18), the changes in the activity caused by the different groups introduced are more conspicuous. In those compounds, due to the changes in the conformation of A/c-lactone ring system or the absence of the double bond in the 2-O bearing ring, the location of 2oxygen group may now be more easily affected by the groups introduced to modify the activity of the compound. Thus 15 and 18 showed no activity, whereas butanoyl 3,4-dihydrosalviandulin E analogue (14) showed a potent antitrypanosomal activity with the IC50 of 0.055 lg/mL (N = 4, SD ± 0.025), with the selectivity index of 1236 (Table 1). Of the three currently used therapeutic drugs used as the control in the present assay, suramin and eflornithine showed rather weak antitrypanosomal activity, implying that the antitrypanosomal activity is not their major therapeutic effect expected: they may cure the disease by some other way. Our present results give positive support to our hypothesis that the antitrypanosomal activity of this series of compounds and the location and the length of alkyl chain of the 2-oxygen function may be closely related. We also consider that 14 can be a possible candidate for an antitrypanosomal drug. Further studies including in vivo tests of the compound 14 in animal models are now in progress. Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (To Y. A.) and funds from the Drugs for Neglected Diseases initiative (DNDi). Our thanks are due to Ms Miyuki Namatame (Kitasato University) for technical assistance.
Figure 3. Aligned stereodrawing of 3 and 11, which were produced by X-ray crystallographic analysis.
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O
O
R
O
O O
H N
(RCO) 2O pyridine
MOMCl Et3 N
O
O
O
H O
O
O H O O 11 DMSO -78 °C (MeCO) 2O
O
products yields (%) R = Et 15 85 n Pr 16 63 Ph 17 70
O
O H O
O
R-N=C=O
HO
O
O
O O
O
H O O products yields (%) R = Me 12 66 Et 13 65 n Pr 14 59
O
R
DMSO (RCO)2 O
O
rt R
O
O O
O
O H O
18 62% O
+
19 24%
O H O
O
22 26%
O
products yields (%) R = Me 19 41 Et 20 26 nPr 21 12
Scheme 3. Preparation of dehydrosalviandulin E (11) analogues.
Table 1 IC50 values of in vitro antitrypanosomal activities (Trypanosoma brucei brucei GUTat 3.1) and of cytotoxicities (MRC-5 cells) of salviandulin E (1), its analogues (2 and 4–22), and currently used drugs, and their ratios. Compounds
Salviandulin E (1) 2 4 5 6 7 8 9 10 11 12 13 14c 15 16 17 18 19 20 21 22 Pentamidine suramina Eflornithinea a b c
IC50 (lg/mL)
Selectivity index (SI)
Antitrypanosomal activity (GUTat 3.1)
Cytotoxicity (MRC-5)
Cytotoxicity IC50/antitrypanosomal activity IC50
0.72 >12.5 0.69 2.39 1.04 0.20 6.08 7.17 1.15 >12.5 7.73 5.33 0.06 >12.5 7.77 10.55 >12.5 4.29 5.56 3.89 >12.5 0.0017 1.58 2.27
0.84 16.17 0.84 1.41 1.05 0.18 44.29 17.27 2.00 NDb >100 63.48 68.00 NDb 66.16 >100 NDb 17.78 22.59 21.56 NDb 5.7 >100 >100
1.17 — 1.22 0.59 1.01 0.90 7.29 2.41 1.73 — >12.9 11.91 1236 — 8.51 >9.5 — 4.14 4.06 5.54 — 3353 >63 >44
Ref. 4a. Not determined. Experimental data for 14: IC50 was 0.055 lg/mL (N = 4, SD ± 0.025).
References and Notes 1. http://www.dndi.org/ 2. Steverding, D. Steverding Parasites & Vectors 2010, 3, 15. and the references cited therein. 3. Fairamb, A. H. Trends Parasitol. 2003, 19, 488. 4. (a) Otoguro, K.; Ishiyama, A.; Namatame, M.; Nishihara, A.; Furusawa, T.; Masuma, R.; Shiomi, K.; Takahashi, Y.; Yamada, H.; Omura, S. J. Antibiot. 2008,
61, 372; (b) Ishiyama, A.; Otoguro, K.; Namatame, M.; Nishihara, A.; Furusawa, T.; Masuma, R.; Shiomi, K.; Takahashi, Y.; Ichimura, M.; Yamada, H.; Omura, S. J. Antibiot. 2008, 61, 627; (c) Otoguro, K.; Iwatsuki, M.; Ishiyama, A.; Namatame, M.; Nishihara-Tukashima, A.; Kiyohara, H.; Hashimoto, T.; Asakawa, Y.; Omura, S.; Yamada, H. Phytochemistry 2011, 72, 2024; (d) Otoguro, K.; Ishiyama, A.; Iwatsuki, M.; Namatame, M.; Nishihara-Tukashima, A.; Kiyohara, H.; Hashimoto, T.; Asakawa, Y.; Omura, S.; Yamada, H. J. Nat. Med. 2012, 66, 377; (e) Otoguro, K.; Iwatsuki, M.; Ishiyama, A.; Namatame, M.; Nishihara-
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6.
7.
8.
9.
Y. Aoyagi et al. / Bioorg. Med. Chem. Lett. 24 (2014) 442–446 Tukashima, A.; Kiyohara, H.; Hashimoto, T.; Asakawa, Y.; Omura, S.; Yamada, H. J. Nat. Med. 2012, 66, 558. and the references cited therein. (a) Esquivel, B.; Dominguez, M.; Hernandez-Ortega, S.; Toscano, R. A.; Rodriguez-Hahn, L. Tetrahedron 1994, 50, 111593; (b) Narukawa, Y.; Hatano, K.; Takeda, T. J. Nat. Med. 2006, 60, 206. (a) Aoyagi, Y.; Yamazaki, A.; Nakatsugawa, C.; Fukaya, H.; Takeya, K.; Kawauchi, S.; Izumi, H. Org. Lett. 2008, 10, 4429; (b) Aoyagi, Y.; Yamazaki, A.; Kato, R.; Tobe, F.; Fukaya, H.; Nishikawa, T.; Nakahashi, A.; Miura, N.; Monde, K.; Takeya, K. Tetrahedron Lett. 2011, 52, 1851. Xu, G.; Peng, L.; Niu, X.; Zhao, Q.; Li, R.; Sun, H. Helv. Chim. Acta 2004, 87, 949. In the present study, 0.018 g of 6,7-dehydrodugesin A (2) was obtained from the aerial parts of this plant (dry weight 18.7 kg). The physical and spectral data of 2 were identical with those reported. To a solution of salviandulin 1 (0.02 g, 0.057 mmol) in CH2Cl2-pyridine (3:1, 2 mL) were added p-bromobenzoyl chloride (0.066 g, 0.30 mmol) and DMAP (0.008 g, 0.0065 mmol) sequentially. The reaction mixture was then stirred at rt for 4 h. Usual work-up and subsequent silica gel column chromatography (CHCl3:MeOH = 10:1) gave the corresponding p-bromobenzoate (3) in 92% yield. Colorless amorphous solid, [a]D: +37.9°(c 0.21, CHCl3), IR(film) m max 1759 (C@O), 1721 (C@O) cm1. HRESIMS: m/z 535.0391 [(M+H)+, calcd for C27H20O7Br, 535.0392]. 1H NMR (pyridine-d5, 500 MHz): d 2.05(3H, s), 2.68(1H, br dd), 3.32(1H, dd, 15.4, 5.3), 3.42(1H, d, 8.4), 4.10(1H, d, 8.4), 5.51(1H, d, 9.1), 5.68(1H, ddd, 10.2, 5.3, 2.0), 6.53(1H, s), 6.72(1H, s), 7.03(1H, d, 9.1), 7.63(1H, s), 7.68(1H, s), 7.74(2H, d, 8.4), 7.99(1H, s), 8.08(2H, d, 8.4). 13C NMR (pyridined5, 125 MHz): d 16.7 (q), 34.2 (t), 47.0 (s), 70.7 (d), 75.6 (t), 76.4 (d), 109.2 (d), 121.5 (s), 122.1 (d), 126.3 (s), 127.7 (s), 128.9 (s), 129.0 (s), 129.0 (s), 129.5 (d), 131.8 (d), 132.3 (d), 133.6 (s), 138.8 (d), 142.5 (d), 145.1 (d), 159.4 (s), 165.1 (s), 168.0 (s), 171.5 (s). Crystallographic data for 3 has been deposited with the Cambridge Crystallographic Data Centre as supplementary publication No. CCDC 966202. Copies of the data can be obtained, free of charge, on application to the Director, CCDC (e-mail: [email protected]). To a MeOH solution (5 mL) of 1 (0.05 g, 0.14 mmol) was added sodium borohydride (0.006 g, 0.16 mmol) at 0°C and the mixture was stirred at the
same temperature for 6 h. Then, H2O (10 mL) was added to the mixture at rt Usual work-up followed by silica gel column chromatography (hexanes: Me2CO = 1.7:1) of the resulting product gave 11 in 80% yield. Colorless needles, mp 119–121 °C, ½a25 D : 125.2 (c 0.11, CHCl3), IR (film): m max 1754 (C@O) cm1, HRESIMS: m/z 355.1163 [(M+H)+, calcd for C20H19O7, 355.1182]. 1H NMR (CDCl3, 500 MHz): d 2.05 (3H, s), 2.24 (1H, ddd, 14.2, 6.6, 5.8), 2.32 (1H, ddd, 14.2, 6.4, 3.7), 2.58 (1H, dd, 17.5, 3.8), 2.71 (1H, dd, 17.5, 6.9), 3.00 (1H, dd, 5.4, 1.6), 3.55 (2H, s), 4.07 (1H, m), 5.56 (1H, d, 9.3), 6.21 (1H, d, 1.0), 6.29 (1H, s), 6.80 (1H, dd, 9.3, 1.5), 7.43 (1H, dd, 1.7, 1.7), 7.52 (1H, d, 0.7). 13C NMR (CDCl3, 125 MHz): d 16.9 (q), 29.6 (t), 36.6 (t), 44.9 (d), 46.2 (s), 65.7 (d), 75.1 (t), 76.3 (d), 108.2 (d), 120.1 (s), 121.4 (d), 125.2 (s), 125.3 (s), 128.9 (d), 136.8 (s), 140.7 (d), 144.6 (d), 158.8 (s), 171.3 (s), 177.7 (s). Crystallographic data for 11 has been deposited with the Cambridge Crystallographic Data Centre as supplementary publication No. CCDC 966201. Copies of the data can be obtained, free of charge, on application to the Director, CCDC (e-mail: [email protected]). 10. Molecular modeling software SYBYL7.3 (Tripos associates, St. Louis, MI, USA) was employed. The X-ray structures obtained were aligned so that A and clactone rings were superimposed to form a common structure by using the database aligned method. 11. In vitro antitrypanosomal activities against T. b. brucei strain GUTat 3.1 were measured as described previously.4 In brief, the strain was cultured in Iscove’s modified Dulbecco’s medium (IMDM) with various supplements and 10% heatinactivated fetal bovine serum at 37 °C, under 5.0% CO2-95% air. Subsequently, 95 ll of the trypanosomes suspension (2.0–2.5 104 trypanosomes/ml) was transferred in a 96-well microtiter plate and 5.0 ll of a test compound solution (dissolved in 5.0% dimethylsulfoxide) was added and incubated for 72 h at 37 °C. Then, 10 ll of the fluorescent dye AlamarBlue was added to each well. After incubation for 3–6 h, the resulting solution was read at 528/20 nm excitation wavelengths and 590/30 nm emission wavelengths by an FLx800 fluorescence microplate reader (Bio-Tek Instruments, Inc. Winooski, VT, USA).The IC50 values were determined using the fluorescent plate reader software (KC-4, Bio-Tek).