Trypanocidal Activity of 2,5-Diphenyloxazoles Isolated from the Roots

Oct 31, 2016 - Eleven 2,5-diphenyloxazole derivatives (1–11), together with six known isoflavonoid derivatives, were isolated from the roots of Oxyt...
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Trypanocidal Activity of 2,5-Diphenyloxazoles Isolated from the Roots of Oxytropis lanata Orkhon Banzragchgarav,† Toshihiro Murata,*,† Gendaram Odontuya,‡ Buyanmandakh Buyankhishig,§ Keisuke Suganuma,⊥,# Bekh-Ochir Davaapurev,§ Noboru Inoue,∥ Javzan Batkhuu,§ and Kenroh Sasaki† †

Department of Pharmacognosy, Tohoku Medical and Pharmaceutical University, 4-1 Komatsushima 4-chome, Aoba-ku, Sendai 981-8558, Japan ‡ Natural Product Chemistry Laboratory, Institute of Chemistry and Chemical Technology, Mongolian Academy of Sciences, 13330 Peace Avenue, The 4th Building of MAS, Ulaanbaatar, Mongolia § School of Engineering and Applied Sciences, National University of Mongolia, POB-617, Ulaanbaatar-46A, Mongolia ⊥ National Research Center for Protozoan Diseases, and #Research Center for Global Agromedicine, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, Hokkaido 080-8555, Japan ∥ Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro, Hokkaido 080-8555, Japan S Supporting Information *

ABSTRACT: Eleven 2,5-diphenyloxazole derivatives (1−11), together with six known isoflavonoid derivatives, were isolated from the roots of Oxytropis lanata. The 2,5-diphenyloxazole (1) obtained proved to be identical to a standard sample used as a scintillator and liquid laser dye. The other oxazole derivatives isolated were found to have one to four hydroxy and/or O-methyl groups in their phenyl rings. Seven of the oxazole derivatives obtained are new (3−9). The inhibitory activity of the isolated compounds was evaluated against Trypanosoma congolense, the causative agent of African trypanosomosis in animals. Oxazoles with di- and trihydroxy groups showed trypanocidal activity, and 2(2′,3′-dihydroxyphenyl)-5-(2″-hydroxyphenyl)oxazole (4) exhibited the most potent inhibitory activity (IC50 1.0 μM). isoflavonoid derivatives. Seven of the oxazoles (3−9) are new, and four (1, 2, 10, and 11) have not previously been identified as natural products. Compound 1 was identified as 2,5-diphenyloxazole, which is a well-known UV fluorescent molecule commonly used as a scintillator and liquid laser dye in applications of physics.8,9 In addition, the spectroscopic features of 2 and 3 were similar to those of oxytrofalcatins A and F, respectively.10 Although oxytrofalcatins A and F have been reported as indoles,10,11 it may be suggested that these oxytrofalcatins are oxazoles.

Oxytropis lanata (Pall.) DC. is a perennial herb belonging to the family Fabaceae. Although some plants in the genus Oxytropis are used as traditional medicinal herbs, others are classified as poisonous plants,1,2 known as “locoweeds”, and contain the phytotoxin swainsonine, which is an intoxicant to livestock and other animals. Seventy-eight species of Oxytropis, including O. lanata, distributed in the territory of Mongolia, have been characterized as traditional Mongolian and Tibetan medicinal plants.3 For instance, O. myriophylla DC. is a popular medicinal plant, which is commonly used to treat bacterial fever, fever from anthrax, bone fractures, bleeding, inflammation, and wounds. O. lanata has also been used for the management of the above-mentioned diseases and symptoms.3 More than 125 chemical compounds have been reported from the genus Oxytropis, including flavonoids, alkaloids, saponins, and lignans.2 Flavonoids are the main constituents and have various biological activities, such as anti-inflammatory and analgesic activities in vivo,4 as well as antitumor,5 antibacterial,6 and antifungal activities.7 In addition, various bioactive alkaloids, including quinolizidines, indolizidines, and quinolones, have been reported in Oxytropis species.2 In this study, 11 2,5-diphenyloxazoles (1−11) were isolated from the roots of O. lanata together with six known © 2016 American Chemical Society and American Society of Pharmacognosy

Received: August 24, 2016 Published: October 31, 2016 2933

DOI: 10.1021/acs.jnatprod.6b00778 J. Nat. Prod. 2016, 79, 2933−2940

129.2 130.7 129.2 125.9

127.4 124.0 129.1 128.6 129.1 124.0

3′ 4′ 5′ 6′

1″ 2″ 3″ 4″ 5″ 6″

2934

a

7.86, 7.52, 7.41, 7.52, 7.86,

m m m m m

m m m m

3″ 2″,4″ 3″ 4″, 6″ 5″

6′ 5′

1′ 2, 2′, 4′

5, 4″, 6″ 1″ 2″ 1″ 5, 2″,4″

2′

3′

NOE

1′

2, 4′, 6′

8.10, m

7.57, 7.57, 7.57, 8.10,

2

HMBC

7.86, s

δH (J in Hz) δC

114.6 148.0 115.8 129.2 119.3 125.3

129.0 130.3 129.0 125.8

158.8 126.5 153.8 126.8 125.8

10.48, s

7.01, d (7.5) 7.22, m 6.97, t (7.5) 7.84, dd (7.5, 1.5)

8.10, dd (8.0, 2.0) 7.55c 7.55c 7.55c 8.10, dd (8.0, 2.0)

7.65, s

δH (J in Hz)

2a

1″

1″ 2″

1″, 5″

4′

4′

HMBC

In DMSO-d6 solution. bIn acetone-d6 solution. cUnclear signal pattern due to overlapping.

OH-2′ OH-5′ or OMe5′ OH-2″ OH-3″ OH-5″

160.2 124.2 150.8 126.8 125.9

δC

2 4 5 1′ 2′

position

1a

Table 1. NMR Spectroscopic Data for Compounds 1−4

4,3″

4″, OH-2″ 3″, 5″ 4″, 6″ 5″

6′ 5′

2′

3′

OH-2″

NOE

δC

116.3 154.6 116.7 129.9 120.8 126.4

158.7 118.2 131.0 118.2

160.3 128.0 149.1 129.8 113.5

7.07, m 7.23, m 7.02, m 7.88, dd (7.5, 1.5)

6.99, m 7.38, t (8.5) 7.65, m

7.64, m

7.69, s

δH (J in Hz)

3b

1″, 2″, 4″, 5″ 2″, 6″ 1″, 3″, 4″, 6″ 5, 2″, 4″

2′, 6′ 1′, 3′ 2, 2′, 4′

2, 4′, 6′

2

HMBC

4″ 3″, 5″ 4″, 6″ 5″

5′ 4′, 6′ 5′

NOE

δC

115.6 154.8 116.8 130.5 120.9 126.6

146.9 118.6 120.6 117.2

160.7 125.4 148.3 111.9 146.4

7.11, br d (8.0) 7.28, m 7.05, m 7.94, dd (7.5, 2.0)

7.02, dd (8.0, 2.0) 6.93, t (8.0) 7.56, dd, (8.0, 2.0)

7.80, s

δH (J in Hz)

4b

1″, 2″, 5″ 2″,6″ 1″ 5, 2″, 4″

2′, 6′ 1′, 3′ 2, 2′

2

HMBC

5″ 4″, 6″ 5″

5′ 4′, 6′ 5′

NOE

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DOI: 10.1021/acs.jnatprod.6b00778 J. Nat. Prod. 2016, 79, 2933−2940

2935

117.6 120.1

150.0 110.9

114.1 154.0 115.9

129.6

119.4

125.3

3′ 4′

5′ 6′

1″ 2″ 3″

4″

5″

6″

a

1′, 2′, 3′ 4′, 5′, 6′ 1″, 2″

10.60, s

1″, 3″, 4″, 6″ 5, 2″, 4″

2″, 3″, 6″

1″, 2″, 5″

2, 2′, 4′

1′, 5′ 2′, 6′

2

HMBC

7.80, dd (8.0, 1.0) 10.49, s 9.25, s

7.00, br t (8.0)

7.26, m

7.03, br d (8.0)

7.38, d (2.5)

6.91, d (8.5) 6.89, dd (8.5, 2.5)

7.74, s

δH (J in Hz)

4, 3″

4, 3′

5″

4″, 6″

3″, 5″

OH-2″, 4″

OH-2′

OH-2′, OH2″

NOE

δC

110.9

149.9

116.2

114.7 146.7 116.6

129.1 125.7

129.1 130.3

148.2 126.6 125.7

158.6 126.6

1″, 2″ 4″, 5″, 6″

8.99, s

2″, 4″, 5″

1″, 2″, 5″ 2″, 5″, 6″

2, 2′, 4′

2, 4′, 6′

2

HMBC

9.77, s

7.22, d (3.0)

6.66, dd (8.5, 3.0)

6.83, d (8.5)

7.53−7.60c 8.07, dd (8.0, 2.0)

8.07, dd (8.0, 2.0) 7.53−7.60c 7.53−7.60c

7.64, s

δH (J in Hz)

6a

In DMSO-d6 solution. bIn acetone-d6 solution. cUnclear signal pattern due to overlapping.

OH-2′ OH-5′ or OMe-5′ OH-2″ OH-3″ OH-5″

146.9 110.4 149.4

5 1′ 2′

δC

158.8 124.4

position

2 4

5a

Table 2. NMR Spectroscopic Data for Compounds 5−8

4″

4, 3″

OH-2″, 4″ 3″,OH5″

6′ 5′

2′

3′

OH-2″

NOE

δC

110.9

150.1

116.8

114.2 146.9 116.7

150.0 110.8

117.7 120.2

147.1 110.5 149.4

158.7 124.5

9.05, br s

9.88, br s

10.46, s 9.27, br s

7.19, d (3.0)

6.68, dd (9.0, 3.0)

6.84, d (9.0)

7.31, d (3.0)

6.91, d (9.0) 6.88, dd (9.0, 3.0)

7.70, s

δH (J in Hz)

7a

4″, 5″, 6″

1″

1′, 2′, 3 4′, 6′

5, 2″

2″

1″, 5″

2, 2′, 4′

5′

2

HMBC

6′

3″

4″

6″

NOE

δC

55.6

111.1

149.9

116.6

114.1 146.9 116.6

152.2 109.2′

117.8 119.4

147.3 110.5 150.5

158.4 124.4

9.02, br s

9.87, br s

10.67, s 3.82, s

7.27, d (3.0)

6.68, dd (9.0,3.0)

6.83, d (9.0)

7.43, d (3.0)

7.01, d (9.0) 7.06, dd (9.0,3.0)

7.72, s

δH (J in Hz)

8a

1″, 2″

3′ 5′

2″, 5″

1″, 2″, 5″ 6″

2

2′, 5′ 2′, 6′

2

HMBC

6″

4, 3″

4′, 6′

OH-5″

OH-2″, 4″ 3″

OMe-5′

OMe-5′

OH-2″

NOE

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Table 3. NMR Spectroscopic Data for Compounds 9−11 9b position

δC

2 4 5 1′ 2′ 3′ 4′

162.2 123.1 151.7 112.0 152.2 119.2 121.8

5′ 6′ 1″ 2″ 3″ 4″

151.5 112.3 128.9 125.7 130.5 130.3

5″ 6″ OH-2′ OH-5′ or OMe5′ OH-2″ OH-3″ OH-5″

130.5 125.7

a

δH (J in Hz)

10b HMBC

7.81, s

2

6.91, d (9.0) 6.97, dd (9.0, 3.0)

5′ 2′

7.47, d (3.0)

2′

7.90, m 7.53, m 7.43, m

5 1″

7.53, m 7.90, m 10.51, s

1″ 5

NOE

δC 161.7 124.7 152.1 missing 151.9 113.6 118.3

3″ 2″

6″ 5″

11a

δH (J in Hz)

HMBC

NOE

7.68, s

2

2″, 6″

7.64, m 7.62, m

131.1 118.5 missing 125.0 129.9 129.4

7.38, m 7.01, m

129.9 125.0

7.51, m 7.85, m

7.85, m 7.51, m 7.41, m

4′ 3′, 5′

4′ 5, 4″, 6″

5, 2″, 4″

4′, 6′ 5′

δC 160.0 124.1 150.9 126.8 125.9 129.2 130.6

4, 3″ 2″, 4″ 3″, 5″

129.2 125.9 128.5 110.6 157.9 115.8

4″, 6″ 4, 5″

130.3 114.9

δH (J in Hz)

HMBC

NOE

7.78, s

2

6″

8.07, m 7.56c 7.56c

2, 4′, 6′ 1′, 5′

3′ 2′

7.56c 8.07, m

1′, 3′ 2, 2′, 4′

6′ 5′

7.21, br s

5, 3″, 4″, 6″

6.80, dt (7.5, 2.0) 7.30, t (7.5) 7.28c

2″, 6″ 1″, 3″ 5

5″, OH3″ 4″, 6″ 4, 5″

9.72, s

2″, 3″, 4″

4″

In DMSO-d6 solution. bIn acetone-d6 solution. cUnclear signal pattern due to overlapping.

Oxazole-type alkaloids are rare natural products, but the oxazole structure is found as a fundamental moiety of certain pharmaceuticals and drug candidate compounds, including aleglitazar and oxaprozin.12 Thus, oxazoles, especially those containing a phenyl moiety, may be effective pharmaceuticals.12 Although data on the biological effects of natural oxazoles are not abundant, there are two reports on the antibacterial activity of texamine and texaline isolated from Amyris texana (Rutaceae).13,14 Recently, it has been reported that the 2,5diphenyl oxazoles, gymnothecaoxazoles A and B, were obtained from Gymnotheca chinensis (Saururaceae), and these compounds have a similar skeleton to the oxazoles isolated herein (1−11).15 The possible inhibitory activity of the isolated oxazoles (1− 11) and isoflavonoid derivatives was determined against Trypanosoma congolense, based on the traditional usage of Oxytropis species for the treatment of infectious diseases. Currently, only a few vaccines and specific medicines are available for protozoan diseases, such as African trypanosomosis, in humans and animals. African trypanosomosis, known as “nagana”, is caused mainly by T. congolense in animals,16 whereas T. brucei causes African sleeping sickness in humans. In turn, T. evansi and T. equiperdum are known to cause the nontsetse-transmitted animal trypanosomal diseases “surra” and “dourine”, respectively. Recently, a trypanosome strain was isolated from a dourine horse in Mongolia and was established as a new reference strain of T. equiperdum.17 Animal husbandry, which is an important sector of the Mongolian economy, is characterized as producing ecologically pure products. Thus, livestock health is important for product quality, and damage to livestock caused by trypanosomoses is a serious problem both for the livelihood of the population and for the national economy. The determination of trypanocidal compounds from the native plants of Mongolia is important from a veterinary

point of view and also contributes to the characterization of unique phytochemical features of the flora.

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RESULTS AND DISCUSSION A MeOH extract of the air-dried roots of O. lanata was suspended in H2O and then fractioned with dichloromethane. The CH2Cl2 extract was subjected to passage over a Sephadex LH-20 column followed by high-performance liquid chromatography, and 11 alkaloids (1−11) and six isoflavonoid derivatives were obtained. 3,9-Dimethyl-10-hydroxypterocarpan,18 vesticarpan,19 (3R)-(−)-arizonicanol A,11,20 5′-methylvestitol,21 odoratin,22 and afromosin23 were identified by comparing their spectroscopic data with those in the literature. Compounds 1−11 were all obtained as colorless powders. Their 1H and 13C NMR spectroscopic features (Tables 1−3) included phenolic resonances in the aromatic regions. Their odd-numbered EIMS data suggested that they contain nitrogen and are alkaloids. The HREIMS analysis of 1 showed a molecular ion [M]+ peak at m/z 211.0832, corresponding to the molecular formula C 15 H 11 NO. In the 1 H NMR spectrum, two sets of unsubstituted aromatic ring resonances (δ 8.10, 2H, m, H-2′ and H-6′; 7.57, 3H, m, H-3′, H-4′, and H-5′; 7.86, 2H, m, H-2″ and H-6″; 7.52, 2H, m, H-3″ and H-5″; 7.41, 1H, m, H-4″) and a singlet proton resonance at δ 7.86 (1H, H-4) were observed. The molecular formula, after allowing for the two phenyl groups, suggested the presence of a 2,5-disubstituted oxazole ring. In the HMBC spectrum, the H-2″ and H-6″ protons showed long-range coupling with the oxygenated carbon at δ 150.8 (C-5) and the H-4, 2′, and 6′ protons showed long-range coupling with the C-2 carbon at δ 160.2. These correlations supported the assignment of 1 as 2,5-diphenyloxazole. A synthesized reference sample of 2,5-diphenyloxazole (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) was purchased, and 2936

DOI: 10.1021/acs.jnatprod.6b00778 J. Nat. Prod. 2016, 79, 2933−2940

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its 1H and 13C NMR spectroscopic data were identical with those of compound 1 (Figures S1 and S2, Supporting Information). Therefore, 1 was identified as 2,5-diphenyloxazole. Some 1H and 13C NMR spectroscopic features of 2−11 were analogous with those of the 2,5-disubstituted oxazole ring of 1. Thus, the resonances of a singlet olefinic proton (7.64−7.86 ppm, H-4) and three olefinic carbons (around 160 ppm, C-2; around 125 ppm, C-4; around 150 ppm, C-5) were observed for all these compounds. Compound 2 showed a molecular formula of C15H11NO2 (HREIMS m/z 237.0796), with one more oxygen atom than 1. A hydroxy proton resonance at δ 10.48 (1H, s, OH-2″) and 2,5disubstituted benzene ring proton resonances (δ 7.01, 1H, d, J = 7.5 Hz, H-3″; 7.22, 1H, m, H-4″; 6.97, 1H, t, J = 7.5 Hz, H5″; 7.84, 1H, dd, J = 7.5, 1.5 Hz, H-6″) were observed in its 1H NMR spectrum, instead of the unsubstituted phenyl moiety resonance of 1. In the NOE spectrum, the correlation between the OH-2″ proton and H-4 (δ 7.65, 1H, s) indicated the presence of a 2″-hydroxyphenyl moiety as ring B. Therefore, 2 was determined as 2-phenyl-5-(2″-hydroxyphenyl)oxazole. Compound 3 showed a [M]+ ion peak in the HREIMS at m/ z 253.0735, corresponding to a molecular formula of C15H11NO3, or two more oxygen atoms than 1, suggesting that 3 has two hydroxy groups in addition to a 2,5diphenyloxazole unit. The o-disubstituted benzene ring 1H and 13C NMR spectroscopic features of 3 were similar to those of 2. Resonances at δ 7.64 (1H, m, H-2′), 6.99 (1H, m, H-4′), 7.38 (1H, t, J = 8.5 Hz, H-5′), and 7.65 (1H, m, H-6′) were observed in the 1H NMR spectrum of 3. These data and the NOE correlations from H-4′ to H-5′ and from H-5′ to H-6′ indicated that 3 has an m-disubstituted benzene moiety as ring A instead of the 2-phenyl moiety of 2. From these data, 3 was established as 2-(3′-hydroxyphenyl)-5-(2″-hydroxyphenyl)oxazole. The 1H and 13C spectroscopic data of 2 and 3 (Table 1) were very similar to those of the reported indole alkaloids oxytrofalcatins A and F, respectively, which were isolated from the roots of Oxytropis falcata (Table S1, Supporting Information).10 Chen et al. assigned the lowest field shifted carbon resonances in the 13C NMR spectra of oxytrofalcatins A and F to amide carbonyl carbons. However, the 13C NMR chemical shifts of typical amide carbonyl carbons of indoles are around 170 ppm.24 Carbon resonances around 160 ppm reasonably may be attributed to the imine carbon of 2,5disubstituted oxazoles.25 Furthermore, the key HMBC longrange correlation between H-6″ and C-5 of 3 demonstrated the presence of an oxazole moiety, and not an indole moiety. Therefore, the oxytrofalcatin alkaloids reported from O. falcata may also be oxazole derivatives. Compounds 4 and 5 were assigned a molecular formula of C15H11NO4 on the basis of their HREIMS data (4, m/z 269.0700 [M]+; 5, m/z 269.0688 [M]+), and both were found to contain three more oxygen atoms than 1. Several NMR spectroscopic features of 4 and 5 were similar to those of 3, except the resonances of ring A. For 4, a set of coupled proton resonances at δ 7.02 (1H, dd, J = 8.0, 2.0, H-4′), 6.93 (1H, t, J = 8.0, H-5′), and 7.56 (1H, dd, J = 8.0, 2.0, H-6′) was observed in its 1H NMR spectrum, which suggested the presence of a 2′,3′dihydroxyphenyl moiety as ring A. The HMBC long-range coupling between the H-6′ proton and C-2 carbon (δ 160.7) and between the H-6″ proton (δ 7.94, 1H, dd, J = 7.5, 2.0) and C-5 carbon (δ 160.7) supported the assignment of rings A and

B in 4, respectively. For 5, an ABX pattern of proton resonances at δ 6.91 (1H, d, J = 8.5, H-3′), 6.89 (1H, dd, J = 8.5, 2.5, H-4′), and 7.38 (1H, d, J = 2.5, H-6′) was observed in its 1H NMR spectrum. The HMBC long-range coupling between the OH-2′ proton (δ 10.49, 1H, s) and C-1′ (δ 110.4), C-2′ (δ 149.4), and C-3′ (δ 117.6) carbons and the NOE correlations between the OH-2′ proton and the H-4 (δ 7.74, 1H, s) and H-3′ protons indicated the presence of a 2′,5′dihydroxyphenyl moiety as ring A. Therefore, 4 and 5 were assigned as 2-(2′,3′-dihydroxyphenyl)-5-(2″-hydroxyphenyl)oxazole and 2-(2′,5′-dihydroxyphenyl)-5-(2″-hydroxyphenyl)oxazole, respectively. The 1H and 13C NMR spectroscopic features of ring B of compounds 6−8 were very similar to each other (Table 2), which suggested that these compounds have different substituent patterns in ring A. In the 1H NMR spectrum of 6, an ABX pattern of proton resonances at δ 6.83 (1H, d, J = 8.5, H-3″), 6.66 (1H, dd, J = 8.5, 3.0, H-4″), and 7.22 (1H, d, J = 3.0, H-6″) and two hydroxy proton resonances at δ 9.77 (1H, s, OH-2″) and 8.99 (1H, s, OH-5″) were observed. The NOE correlations from H-4 (δ 7.64, 1H, s) to OH-2″, OH-2″ to H3″, H-3″ to H-4″, and H-4″ to OH-5″ demonstrated that ring B of 6 is a 2″,5″-dihydroxyphenyl moiety. A set of unsubstituted aromatic ring resonances (δ 8.07, 2H, dd, J = 8.0, 2.0, H-2′ and 6′; 7.53−7.60, 3H, overlapping, H-3′, 4′, and 5′) indicated that 6 has a phenyl group at C-2 as ring A. Compound 6 showed a [M]+ ion peak in the HREIMS at m/z 253.0749, corresponding to a molecular formula of C15H11NO3. These data supported the assignment of 6 as 2-phenyl-5-(2″,5″-dihydroxyphenyl)oxazole. Compound 7 was assigned a molecular formula of C15H11NO5 on the basis of its HREIMS (m/z 285.0636 [M]+) and thus has two oxygen atoms more than 6. The 1H and 13C NMR resonances of ring A of 7 were very close to those of 5, which indicated the presence of a 2′,5′dihydroxyphenyl moiety of 7. The HMBC long-range coupling between the H-6′ proton (δ 7.31, 1H, d, J = 3.0) and C-2 carbon (δ 158.7) and between the H-6″ proton (δ 7.19, 1H, d, J = 3.0) and C-5 carbon (δ 147.1) supported the assignment of rings A and B in 7, respectively. Therefore, 7 was determined as 2-(2′,5′-dihydroxyphenyl)-5-(2″,5″-dihydroxyphenyl)oxazole. Compound 8 was assigned a molecular formula of C16H13NO5 from its HREIMS data (m/z 299.0805 [M]+) and thus contains one more CH2 than 7, corresponding to an O-methyl substituent. The 1H and 13C NMR resonances of 8 were closely comparable to those of 7, except for the presence of an O-methyl moiety (δ 3.82, 3H, s and δ 55.6, OMe-5′). The O-methyl protons showed long-range coupling with C-5′ (δ 152.2) of ring A. The NOE correlations from the O-methyl proton to H-4′ (δ 7.06, 1H, dd, J = 9.0, 3.0 Hz) and H-6′ (δ 7.43, 1H, d, J = 3.0 Hz) suggested that the O-methyl group of 8 is substituted at C-5′ in place of the hydroxy group of 7. Thus, 8 was determined as 2-(2′-hydroxy-5′-O-methylphenyl)-5(2″,5″-dihydroxyphenyl)oxazole. Compound 9 was assigned the molecular formula C15H11NO3, as determined from its [M]+ peak at m/z 253.0743. A set of unsubstituted aromatic ring resonances (δ 7.90, 2H, m, H-2″ and H-6″; 7.53, 2H, m, H-3″ and H-5″; 7.43, 1H, m, H-4″) and an ABX pattern of proton resonances at δ 6.91 (1H, d, J = 9.0, H-3′), 6.97 (1H, dd, J = 9.0, 3.0, H-4′), and 7.47 (1H, d, J = 3.0, H-6′) were observed in the 1H NMR spectrum (Table 3). In the HMBC spectrum, the H-2″ and H6″ protons showed long-range coupling with the C-5 (δ 151.7) 2937

DOI: 10.1021/acs.jnatprod.6b00778 J. Nat. Prod. 2016, 79, 2933−2940

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carbon, which demonstrated that ring A is a 2′,5′-dihydroxyphenyl moiety and ring B an unsubstituted phenyl moiety. Therefore, 9 was established as 2-(2′,5′-dihydroxyphenyl)-5phenyloxazole. Compounds 10 and 11 were both assigned a molecular formula of C15H11NO2 on the basis of their HREIMS results (10, m/z 237.0780 [M]+; 11, m/z 237.0780 [M]+), identical to that of 2. For 10, a set of unsubstituted aromatic ring resonances (δ 7.85, 2H, m, H-2″ and H-6″; 7.51, 2H, m, H-3″ and H-5″; 7.41, 1H, m, H-4″), similar to 9, suggested that ring B is an unsubstituted phenyl moiety. The HMBC correlations from H-2″ and H-6″ to C-5 and the NOE correlations from H4 to H-2″ and H-6″ supported the conclusion of ring B. The 1′,2′-disubstituted benzene ring proton resonances (δ 7.64, 1H, m, H-3′; 7.62, 1H, m, H-4′; 7.38, 1H, m, H-5′; 7.01, 1H, m, H6′) were observed in the 1H NMR spectrum of 10 and demonstrated that 10 is 2-(2′-hydroxyphenyl)-5-phenyloxazole. For 11, ring A was found to be an unsubstituted phenyl moiety, similar to 1, 2, and 6. The resonances of an m-disubstituted benzene moiety [δ 7.21 (1H, br s, H-2″), 6.80 (1H, dt, J = 7.5, 2.0 Hz, H-4″), 7.30 (1H, t, J = 7.5 Hz, H-5″), and 7.28 (overlapping, H-6″)] were also observed in the 1H NMR of 11. In the HMBC spectrum, H-4, H-2′, and H-6′ showed longrange couplings with C-2 (δ 160.0), and H-2″ and H-6″ with C5 (δ 150.9). From these data, 11 was proposed as 2-phenyl-5(3″-hydroxyphenyl)oxazole. The synthesized compounds 1, 2, 10, and 11 had been reported in previous studies.25−27 However, in the present study, these oxazoles were isolated as natural products for the first time. The inhibitory activities against T. congolense of the isolated oxazoles (1−11) and 3,9-dimethyl-10-hydroxypterocarpan, (3R)-(−)-arizonicanol A, odoratin, and afromosin were determined, and the results are shown in Table 4. Pentamidine

compound

IC50 (μM) 1.0 12.2 12.1 14.8 6.0 4.1 0.17 0.11

EXPERIMENTAL SECTION

General Experimental Procedures. UV spectra were recorded with a Shimadzu MPS-2450 (Shimadzu, Kyoto, Japan). The 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded using a JEOL JNM-AL400 FT-NMR spectrometer (JEOL, Tokyo, Japan), and the chemical shifts are reported as δ values with tetramethylsilane as internal standard (measured in DMSO-d6 and acetone-d6). Inversedetected heteronuclear correlations were measured using HMQC (optimized for 1JC−H = 145 Hz) and HMBC (optimized for nJC−H = 8 Hz) pulse sequences with a pulsed-field gradient. The HREIMS data were obtained using a JEOL JMS700 mass spectrometer (JEOL). Preparative HPLC was performed using a JASCO 2089 instrument with UV detection at 210 nm (JASCO), using the following columns: TSKgel ODS-120T (Tosoh, Tokyo, Japan, 21.5 × 300 mm), Develosil C30-UG-5 (Nomura Chemical, Aichi, Japan, 20 × 250 mm), Cosmosil 5C18-AR-II (Nacalai Tesque, Kyoto, Japan, 20 × 250 mm), and Mightysil RP-18 GP (Kanto Chemical, Tokyo, Japan, 10 × 250 mm). Plant Material. Oxytropis lanata was collected in July 2012, in the territory of Altanbulag Soum, N 47°34.202′; E 106°16.155′, and at an altitude of 1354 m, Tuv Province, Mongolia. A voucher specimen was deposited at the herbarium of National University of Mongolia (49.15.25.12A). Prof. Chinbat Sanchir, Institute of General and Experimental Biology, Mongolian Academy of Sciences, identified the plant species. Extraction and Isolation. The air-dried roots of O. lanata (127 g) were extracted with MeOH (500 mL × 3) at room temperature for 5 days, and after filtration the MeOH extract was taken to dryness under reduced pressure to yield 10.1 g of a MeOH extract. The MeOH extract was suspended in H2O and then fractionated with CH2Cl2, and a CH2Cl2 extract (3.5 g) and an aqueous extract (6.1 g) were obtained. The CH2Cl2 extract was fractionated over a Sephadex LH-20 column (volume 166 mL) using methanol and CH2Cl2 mixtures (3:2 → 1:1) as solvents to afford 12 fractions (OLD-1−12). Compounds 1 (2.4 mg) and 9 (2.0 mg) were isolated from fraction OLD-5 (674.6 mg) using HPLC [columns: ODS-120T, mobile phase: CH3CN−H2O (7:3, v/v) containing 0.2% TFA; 5C18-AR-II, CH3CN−H2O (7:3, v/v) containing 0.2% TFA; RP-18 GP, CH3CN−H2O (3:2, v/v) containing 0.2% TFA]. Compounds 2 (0.8 mg), 3 (25.4 mg), 4 (23.3 mg), 5 (27.1 mg), 6 (64.5 mg), 7 (7.2 mg), 8 (5.8 mg), 10 (2.2 mg), 11 (8.9 mg), 3,9-dimethyl-10-hydroxypterocarpan (25.1 mg), vesticarpan (1.4 mg), (3R)-(−)-arizonicanol A (13.5 mg), 5′-methylvestitol (0.7 mg), odoratin (14.8 mg), and afromosin (7.0 mg) were isolated from fractions OLD-6 (665.4 mg) and OLD-7 (383.2 mg) using HPLC (columns: ODS-120T, mobile phase: CH3CN−H2O (2:3, v/v) containing 0.2% TFA; C30-UG-5, CH3CN−H2O (1:1, v/v) containing with 0.2% TFA; 5C18-AR-II, CH3CN−H2O (7:15, v/v) containing 0.2% TFA; RP-18 GP, CH3CN−H2O (1:1, v/v) containing 0.2% TFA). 2,5-Diphenyloxazole (1): colorless powder; UV (MeOH) λmax (log ε) 304 (4.46) nm; 1H NMR (DMSO-d6, 400 MHz), see Table 1; 13C NMR (DMSO-d6, 100 MHz), see Table 1; HREIMS m/z 221.0832 [M]+ (calcd for C15H11NO, 221.0841). 2-Phenyl-5-(2″-hydroxyphenyl)oxazole (2): colorless powder; UV (MeOH) λmax (log ε) 327 (4.02) nm; 1H NMR (DMSO-d6, 400 MHz), see Table 1; 13C NMR (DMSO-d6, 100 MHz), see Table 1; HREIMS m/z 237.0796 [M]+ (calcd for C15H11NO2, 237.0790). 2-(3′-Hydroxyphenyl)-5-(2″-hydroxyphenyl)oxazole (3): colorless powder; UV (MeOH) λmax (log ε) 329 (4.43) nm; 1H NMR (acetoned6, 400 MHz), see Table 1; 13C NMR (acetone-d6, 100 MHz), see Table 1; HREIMS m/z 253.0735 [M]+ (calcd for C15H11NO3, 253.07393). 2-(2′,3′-Dihydroxyphenyl)-5-(2″-hydroxyphenyl)oxazole (4): colorless powder; UV (MeOH) λmax (log ε) 333 (4.44) nm; 1H NMR (acetone-d6, 400 MHz), see Table 1; 13C NMR (acetone-d6, 100 MHz), see Table 1; HREIMS m/z 269.0700 [M]+ (calcd for C15H11NO4, 269.0688). 2-(2′,5′-Dihydroxyphenyl)-5-(2″-hydroxyphenyl)oxazole (5): colorless powder; UV (MeOH) λmax (log ε) 350 (4.46) nm; 1H NMR (DMSO-d6, 400 MHz), see Table 2; 13C NMR (DMSO-d6, 100

Table 4. Evaluation of Inhibitory Activity against T. congolense for Compounds Isolated from O. lanataa 4 5 6 7 8 (3R)-(−)-arizonicanol A pentamidine16 diminazene16

Article

a Compounds 1−3, 9−11, 3,9-dimethyl-10-hydroxypterocarpan, odoratin, and afromosin were inactive at 20 μM. The treatment was replicated three times for each concentration.

(IC50 0.17 μM) and diminazene (IC50 0.11 μM) were used as positive controls. Oxazoles with dihydroxyphenyl moieties that have a 5′,6′-dihydroxyphenyl unit (4), or a 2′,5′-dihydroxyphenyl unit (5) as ring A, or a 2″,5″-dihydroxyphenyl unit as ring B (6−8) showed moderate inhibitory activities (IC50 1.0− 14.8 μM). An isoflavonoid derivative, (3R)-(−)-arizonicanol A, also showed activity (IC50 4.1 μM). Compound 4 showed the most potent inhibitory activity (IC50 1.0 μM), and the active oxazoles contained more than two hydroxy groups in their phenyl rings. These results suggest that hydroxy group substituents in 2,5-diphenyloxazoles are important factors that contribute to trypanocidal activity. 2938

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MHz), see Table 2; HREIMS m/z 269.0688 [M]+ (calcd for C15H11NO4, 269.0688). 2-Phenyl-5-(2″,5″-dihydroxyphenyl)oxazole (6): colorless powder; UV (MeOH) λmax (log ε) 344 (4.17) nm; 1H NMR (DMSO-d6, 400 MHz), see Table 2; 13C NMR (DMSO-d6, 100 MHz), see Table 2; HREIMS m/z 253.0749 [M]+ (calcd for C15H11NO3, 253.0739). 2-(2′,5′-Dihydroxyphenyl)-5-(2″,5″-dihydroxyphenyl)oxazole (7): colorless powder; UV (MeOH) λmax (log ε) 357 (3.67) nm; 1H NMR (DMSO-d6, 400 MHz), see Table 2; 13C NMR (DMSO-d6, 100 MHz), see Table 2; HREIMS m/z 285.0636 [M]+ (calcd for C15H11NO5, 285.0637). 2-(2′-Hydroxy-5′-O-methylphenyl)-5-(2″,5″-dihydroxyphenyl)oxazole (8): colorless powder; UV (MeOH) λmax (log ε) 356 (4.24) nm; 1H NMR (DMSO-d6, 400 MHz), see Table 2; 13C NMR (DMSO-d6, 100 MHz), see Table 2; HREIMS m/z 299.0805 [M]+ (calcd for C16H13NO5, 299.0794). 2-(2′,5′-Dihydroxyphenyl)-5-phenyloxazole (9): colorless powder; UV (MeOH) λmax (log ε) 348 (3.89) nm; 1H NMR (acetone-d6, 400 MHz), see Table 3; 13C NMR (acetone-d6, 100 MHz), see Table 3; HREIMS m/z 253.0743 [M]+ (calcd for C15H11NO3, 253.0739). 2-(2′-Hydroxyphenyl)-5-phenyloxazole (10): colorless powder; UV (MeOH) λmax (log ε) 305 (4.32) nm; 1H NMR (acetone-d6, 400 MHz), see Table 3; 13C NMR (acetone-d6, 100 MHz), see Table 3; HREIMS m/z 237.0780 [M]+ (calcd for C15H11NO2, 237.0790). 2-Phenyl-5-(3″-hydroxyphenyl)oxazole (11): colorless powder; UV (MeOH) λmax (log ε) 306 (4.52) nm; 1H NMR (DMSO-d6, 400 MHz), see Table 3; 13C NMR (DMSO-d6, 100 MHz), see Table 3; HREIMS m/z 237.0780 [M]+ calcd for C15H11NO2, 237.0790). Evaluation of Trypanocidal Activity. To evaluate trypanocidal activity, the bloodstream form of T. congolense IL3000 strain was used, according to a previous report.16 T. congolense IL3000 was cultivated in Hirumi’s modified Iscove’s medium (HMI)-9, which was prepared by the reported method.28 The final concentrations of compounds 1−11, 3,9-dimethyl-10-hydroxypterocarpan, (3R)-(−)-arizonicanol A, odoratin, and afromosin in the assay ranged from 25 μg/mL to 1.6 ng/mL by 5-fold serial dilution. After the trypanosomes were incubated with chemicals for 72 h, CellTiter-Glo luminescent cell viability assay reagent (Promega Japan, Tokyo, Japan) was added to evaluate intracellular ATP concentration. The plate was shaken using an MS3 basic plate shaker (500 shakes/min for 2 min) (Ika Japan K.K., Osaka, Japan) and then incubated for another 10 min at room temperature. The plate was read using a GloMax-Multi+ detection system plate reader (Promega Japan). The IC50 value of each reference compound was calculated by plotting the percent inhibition (0% inhibition = the luminescence of a trypanosome culture well without any chemicals) against log concentration in GraphPad Prism 5 software (GraphPad Software, Inc.). Pentamidine and diminazene (Sigma-Aldrich) were used as positive controls.

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ACKNOWLEDGMENTS We thank Ms. Y. Matsushita, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, for assistance with the trypanocidal activity measurements, and Mr. S. Sato and Mr. T. Matsuki, Tohoku Medical and Pharmaceutical University, for assistance with the MS measurements. This work was supported by the JICA M-JEED project and a grant from JSPS Kakenhi (grant number JP26860068). This work was partially supported by grants for Research Support Year at the National University of Mongolia, the Kanno Foundation of Japan, the Cooperative Research Grant (28-joint-12) of National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, and AMED/JICA SATREPS.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00778.

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Article

Figures S1 and S2, Table S1, the NMR spectroscopy data for 1−11, including 1H NMR, 13C NMR, 1H−1H COSY, HMQC, and HMBC, and HREIMS data (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel: +81 22 727 0086. Fax: +81 22 727 0220. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 2939

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