Synthesis and biological activity of furostanic analogues of brassinosteroids bearing the 5α-hydroxy-6-oxo moiety

Synthesis and biological activity of furostanic analogues of brassinosteroids bearing the 5α-hydroxy-6-oxo moiety

s t e r o i d s 7 2 ( 2 0 0 7 ) 955–959 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/steroids Synthesis and biologi...

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s t e r o i d s 7 2 ( 2 0 0 7 ) 955–959

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/steroids

Synthesis and biological activity of furostanic analogues of brassinosteroids bearing the 5␣-hydroxy-6-oxo moiety ´ M. Mendez-Stivalet a , Margarita Romero-Avila a , Guadalupe de Dios-Bravo b , Jose Rogelio Rodr´ıguez-Sotres a , Martin A. Iglesias-Arteaga a,∗ a

´ Facultad de Qu´ımica, Universidad Nacional Autonoma de M´exico, Ciudad Universitaria, 04510 M´exico, D.F., Mexico ´ ´ San Isidro 151, Universidad Autonoma de la Ciudad de M´exico, Plantel San Lorenzo Tezonco, Prolongacion 09790 M´exico D.F., Mexico b

a r t i c l e

i n f o

a b s t r a c t

Article history:

Two furostanic analogues of brassinosteroids bearing the 5␣-hydroxy-6-oxo moiety were

Received 22 February 2007

synthesized and their biological activity studied using the bean second internode elongation

Received in revised form

test. One of the compounds produced significant stimulation at doses of 2.5 and 5 ng/plant.

21 July 2007

© 2007 Elsevier Inc. All rights reserved.

Accepted 17 August 2007 Published on line 23 August 2007 Keywords: Furostanic analogues of brassinosteroids Synthesis Plant growth stimulation

1.

Introduction

Brassinosteroids (Bs) are potent plant growth promoters. In addition to more than 70 naturally occurring brassinosteroids [i.e. brassinolide (1)] (Fig. 1) a wide variety of synthetic analogues of brassinosteroids (ABs) has been reported [1–10]. Although the structural requirements for a good biological activity have been clearly recognized, a number of compounds bearing slight to drastic structural modifications are potent plant growth promoters. In particular, some ABs bearing the 5␣-hydroxy-6-oxo moiety like 3 have shown high plant growth promoting activity [7]. As a part of our program on the synthesis of bioactive steroids we have reported the synthesis of some furostanic ABs (i.e. compound 4, Fig. 1) [8–10]. Herein,



we report on the synthesis and biological activity of two new furostanic analogues of brassinosteroids bearing the 5␣hydroxy-6-oxo moiety.

2.

Experimental

2.1.

General

Reactions were monitored by TLC on ALUGRAM® SIL G/UV254 plates from MACHEREY-NAGEL. Chromatographic plates were sprayed with a 1% solution of vanillin in 50% HClO4 and heated until color developed. NMR spectra were recorded in a Varian Unity INOVA 300 MHz spectrometer using TMS for 1 H or the solvent signal (CDCl3 ) for 13 C as reference. Mass spectra were recorded in a JEOL SX-102-A spectrometer. Melting points

Corresponding author. Tel.: +52 55 5622 3704; fax: +52 55 5622 3722. E-mail address: [email protected] (M.A. Iglesias-Arteaga). 0039-128X/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2007.08.007

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s t e r o i d s 7 2 ( 2 0 0 7 ) 955–959

Fig. 1 – Some naturally occurring brassinosteroids and synthetic analogues.

were measured on a Melt-Temp II equipment and are uncorrected. Solvents (THF, pyridine, CH2 Cl2 ) were dried according to established procedures [11]. (22R)-Furost-5-en-3␤-ol acetate (7b) was synthesized as described by Chausuancharoen et al. [12].

2.2.

Synthetic procedures

2.2.1.

(22R)-Furostan-3ˇ,5˛,6ˇ-triol 3-monoacetate (8)

MCPBA (345 mg, 1.4 mmol) was added to a solution of 7b (441 mg, 1 mmol) in CH2 Cl2 (15 ml) and the mixture was stirred for 40 min at room temperature. A solution of Na2 SO3 (10%, 2 ml) was added and the mixture was stirred for 15 min and cooled in an ice bath before addition of acetone (5 ml) and a solution of 1.5 ml of 70% HClO4 in 3 ml of water. The mixture was stirred for 1 h, ethyl acetate (100 ml) was added, and the organic layer was washed with saturated NaCl solution (3 × 15 ml), 10% Na2 CO3 solution (10 × 15 ml) and water (2 × 15 ml), dried (anh. Na2 SO4 ) and evaporated to afford the monoacetylated triol 8 (432 mg, 91%) as a white solid. m.p. 160–162 ◦ C (from ethyl acetate/hexane). 1 H NMR (300 MHz) ı ppm 5.20–5.08 (m, 1H, H-3), 4.29 (dt, J = 7.82, 7.78, 5.09 Hz, 1H, H16), 3.53 (m, 1H, H-6), 3.34–3.26 (m, 1H, H-22), 2.05 (s, 3H, CH3 acetate), 1.20 (s, 3H, H-19), 0.99 (d, J = 6.71 Hz, 3H, H-21), 0.88 (d, J = 6.59 Hz, 6H, H-26 and H-27), 0.81 (s, 3H, H-18). 13 C NMR (75.5 MHz) ı ppm 170.91, 90.44, 83.03, 75.99, 75.63, 71.14, 65.29, 60.38, 56.11, 45.43, 41.07, 39.58, 38.40, 37.89, 37.00, 35.81, 34.80, 32.09, 31.36, 29.97, 28.23, 26.63, 22.55, 22.48, 21.42, 20.68, 19.02, 16.64, 14.17. MS (70 eV) 477 (M+ +1), 405, 347, 311, 269, 251, 122, 81, 69, 55.

2.2.2.

(22R)-3ˇ-Acetoxy-5˛-hydroxy-furostan-6-one (9)

A solution of 8 (407 mg, 0.861 mmol) in CH2 Cl2 (5 ml) was added to a stirred suspension of PCC (278 mg, 1.29 mmol) in CH2 Cl2 (5 ml) and the mixture was stirred for 2.5 h. Ethyl ether (25 ml) was added, and the mixture was filtered through a celite pad. The celite pad was washed with CH2 Cl2 (2 × 20 ml) and the collected fractions were evaporated to afford the ketol 9 (335 mg, 87%). m.p. 184–186 ◦ C (from ethyl acetate/hexane). 1 H NMR (300 MHz) ı ppm 5.15–4.88 (m, 1H, H-3), 4.33–4.23 (m, 1H, H-16), 3.33–3.24 (m, 1H, H-22), 2.75 (dd, J = 11.60, 11.60 Hz, 1H, H-7ax. ), 1.98 (s, 3H, CH3 acetate), 0.98 (d, J = 6.41 Hz, 3H, H-21), 0.86 (d, J = 6.37 Hz, 6H, H-26 and H-27), 0.81 (s, 3H, H-18), 0.76 (s, 3H, H-19). 13 C NMR (75.5 MHz) ı ppm 212.00, 171.06, 90.42, 82.76, 80.10, 70.63, 65.12, 56.46, 44.26, 42.41, 41.69, 41.44, 39.19, 37.85, 36.88, 35.77, 32.29, 31.90, 31.32, 29.48, 28.20, 26.20, 22.52, 22.45, 21.33, 20.96, 18.97, 16.46, 13.92. MS (70 eV) 475 (M+ +1), 414, 405, 296, 267, 239, 161, 147, 121, 93, 81, 69, 55.

2.2.3.

(22R)-3ˇ,5˛-Dihydroxy-furostan-6-one (10a)

A suspension of 9 (947 mg, 2.002 mmol) in a saturated K2 CO3 methanolic solution (15 ml) was stirred at 40 ◦ C for 30 min. The mixture was cooled, poured in ice, filtered and the solid obtained was dried in vacuum to afford the dihydroxylated furostanone 10a (860 mg, 99.4%). m.p. 196–198 ◦ C. 1 H NMR (300 MHz) ı ppm 4.33–4.24 (m, 1H, H-16), 4.07–3.95 (m, 1H, H3), 3.34–3.24 (m, H-22), 2.75 (dd, J = 11.37, 11.37 Hz, 1H, H-7ax. ), 0.99 (d, J = 6.54 Hz, 3H, H-21), 0.87 (d, J = 6.50 Hz, 6H, H-26 and H-27), 0.79 (s, 3H, H-18), 0.76 (s, 3H, H-19). 13 C NMR (75.5 MHz) ı ppm 213.06, 90.44, 82.79, 80.38, 67.46, 65.18, 56.54, 44.37, 42.43, 41.81, 41.50, 39.26, 37.89, 37.09, 36.97, 35.80, 31.94, 31.36, 30.01,

s t e r o i d s 7 2 ( 2 0 0 7 ) 955–959

957

Scheme 1 – Synthetic procedures: reagents and conditions. (a) NaBH3 CN, AcOH; (b) TsCl, pyr; (c) LiAlH4 , THF reflux; (d) Ac2 O, pyr; (e) i MCPBA, CH2 Cl2 , ii acetone, H2 O, HClO4 ; (f) PCC, CH2 Cl2 ; (g) K2 CO3 , CH3 OH; (h) Li2 CO3 , LiBr, DMF reflux; (i) OsO4 , NMMO, THF, H2 O.

29.81, 28.24, 22.54, 22.48, 21.07, 19.15, 19.02, 16.50, 14.07. MS (70 eV) 432 (M+ ), 361, 303, 285, 257, 161, 122, 107, 81, 55, 43. HRMS (FAB), found 433.3323, estimated for C27 H45 O4 (MH+) 433.3318.

2.2.4.

(22R)-5˛-Hydroxy-furost-2-en-6-one (11)

Tosyl chloride (633 mg, 3.319 mmol) was added to a solution of 10a (700 mg, 1.628 mmol) in pyridine (3.5 ml) and the mixture was stirred for 24 h at room temperature before addition of ice and extraction with ethyl acetate (5 × 50 ml). The organic layer was washed with water (5 × 50 ml), dried (anh. Na2 SO4 ) and evaporated. The residue was dissolved in DMF (17 ml), and refluxed for 3 h with LiBr (1.5848 g, 17.96 mmol) and Li2 CO3 (1.3207 g, 17.96 mmol). The mixture was cooled in an ice bath before carefully addition of 10% HCl (100 ml). The solid was filtered off, washed with plenty of water and dried in vacuum to afford the unsaturated ketol 11 (581 mg, 76%). m.p. 100–102 ◦ C (from ethyl acetate/hexane). 1 H NMR (300 MHz) ı ppm 5.80–5.47

(m, 2H, H-2 and H-3), 4.34–4.26 (m, 1H, H-16), 3.34–3.26 (m, H-22), 1.00 (d, J = 6.70 Hz, 3H, H-21), 0.88 (d, J = 6.52 Hz, 6H, H26 and H-27), 0.79 (s, 3H, H-18), 0.72 (s, 3H, H-19). 13 C NMR (75.5 MHz) ı ppm 210.83, 125.44, 122.36, 90.45, 82.73, 77.91, 65.18, 56.63, 45.23, 42.63, 42.25, 41.26, 39.22, 37.91, 37.02, 35.81, 34.56, 31.93, 31.35, 30.14, 28.23, 22.54, 22.47, 20.64, 19.00, 16.38, 14.60. MS (70 eV) 414 (M+ ), 381, 343, 285, 267, 239, 161, 147, 121, 93, 81, 55, 43.

2.2.5.

(22R)-2˛,3˛,5˛-Trihydroxy-furostan-6-one (12)

A solution of OsO4 in tert-butanol (0.39 ml, ca. 12.5 mg/ml) and NMMO (243 mg) were added to a solution of the unsaturated ketol 11 (250 mg, 0.603 mmol) in THF (5 ml) and water (0.5 ml). The mixture was stirred for 24 h, a solution of Na2 SO3 (218 mg) in water (0.5 ml) was added, and the mixture was stirred for 3 h before addition of CH2 Cl2 (100 ml). The organic layer was washed with saturated NaCl solution (5 × 20 ml), dried (anh. Na2 SO4 ) and evaporated. The residue was puri-

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fied by flash chromatography using ethyl acetate:hexane 1:1 to afford the trihydroxylated furostanone 12 (116 mg, 42.9%). m.p. 200–202 ◦ C (from ethyl acetate/hexane). 1 H NMR (300 MHz) ı ppm 4.29 (dt, J = 7.68, 7.52, 4.93 Hz, 1H, H-16), 4.18–4.14 (m, 1H, H-2), 3.92–3.84 (m, 1H, H-3), 3.42–3.17 (m, 1H, H-22), 2.70 (dd, J = 12.46, 12.46 Hz, 1H, H-7ax. ), 0.99 (d, J = 6.70 Hz, 3H, H-21), 0.87 (d, J = 6.59 Hz, 6H, H-26 and H-27), 0.77 (s, 3H, H-18), 0.76 (s, 3H, H-19). 13 C NMR (75.5 MHz) ı ppm 210.84, 90.44, 82.76, 79.24, 69.83, 67.71, 65.09, 56.45, 45.36, 44.59, 41.45, 39.18, 37.87, 36.71, 35.79, 34.27, 31.90, 31.34, 30.06, 28.23, 22.53, 22.47, 20.78, 18.99, 16.46, 14.80. MS (70 eV) 449 (M+ +1), 377, 319, 301, 283, 121, 107, 93, 81, 55. HRMS (FAB), found 447.3118, estimated for C27 H43 O5 (M+ −1) 447.3110.

2.3.

Biological assays

Modified second internode of bean test [13,14]. Seeds of bean (Phaseolus vulgaris L., cv. Pinto) were germinated for 2 days, selected for uniformity from a large population of seedlings and then were planted into pots containing perlite and half strength Hoagland’s solution. The pots were placed in a temperature-controlled greenhouse chamber (25–27 ◦ C, photoperiod 16 h/8 h). Groups of 10 7-day-old plants with second internodes 2 mm long were each treated with different concentrations of tested compounds in lanoline (2 ␮l). The compounds were applied to the sear leaf after the removal of bract from the base of the second internode. The group of control plants was treated with lanoline. The lengths of the second internodes were measured after 5 days and the difference in length between treated and control plants provided a measure of activity. Each experiment was repeated at least three times with new seed lots. The results of repetitions for each compound were analyzed together using Kruskal–Wallis One Way Analysis of Variance on Ranks followed by multiple comparisons with the Student–Newman–Keuls Method to determine the statistical significance of the differences observed between doses. A set of plants treated with lanoline (zero dose) was present on each experiment and included in the statistical analysis. Because dose response curves for the tested compounds were not run in parallel, the sets of control plants (lanolin only) included for each compound were kept separated. To obtain an estimate of a half-stimulatory dose, dose–response curves were approximated to hyperbolas, just as a mean for interpolation. This is an empirical procedure, since the complexity of the physiological phenomena involved does not allow us to apply a particular model at this point.

3.

tion mixture afforded the monoacetylated triol 8. Oxidation of 8 with PCC yielded the ␣-hydroxyketone 9 which was treated with K2 CO3 in methanol to afford the dihydroxylated furostanone 10a. Treatment of 10a with tosyl chloride in pyridine led to the tosylate 10b, which was used in the next step without further purification. Treatment of the tosylate 10b with LiBr and Li2 CO3 in refluxing DMF afforded the unsaturated ketol 11, which was dihydroxylated using the standard OsO4 /NMMO procedure to afford the trihydroxylated furostenone 12 (Scheme 1).

3.1.

Biological activity

The tested compounds, homobrassinolide (2), 10a and 12 dissolved in lanoline (2 ␮l) were applied to pinto bean (P. vulgaris L.) plants in different amounts (2.5, 5, 10 and 15 ng/plant). Results are compared to control plants treated with 2 ␮l of lanoline (Fig. 2). The dihydroxylated ketone 10a produced no significant elongation of the second bean internode at the doses tested, however, the trihydroxylated ketone 12 induced significant stimulation at the two lower doses tested (2.5 and 5 ng; p < 0.01 on a Kruskal–Wallis One Way Analysis of Variance on Ranks). Interestingly, at higher doses the stimulating effect disappears. As expected, homobrassinolide (2) was effective in all the range tested. Internode elongation produced by compound 12 was comparable to the elongation observed with homobrassinolide (2), but only in the lower concentration range. When taking into consideration only the concentration range where stimulation was observed for compound 12, interpolated half-stimulatory doses for homobrassinolide (2) and compound 12 gave similar estimates, in the range of 2–6 ng/plant. The loss of effectiveness observed for compound 12 at higher doses may result from the complex response of plants to Bs. Antagonizing effects of Bs have been reported on wheat and lotus seedlings [18,19], though, in the concentration range tested here, the effect of homobrassinolide (2) was of only stimulatory (Fig. 2).

Results and discussion

Reductive opening of the spiroketal system of diosgenin acetate (5) afforded the monoacetylated furostenol (6a). The 22R configuration has been previously verified in a NOE difference experiment [10]. Treatment of 6a with TsCl in pyridine followed by reduction with LiAlH4 in refluxing THF produced the furostenol 7a, which was acetylated to afford 7b identical as previously described by Chausuancharoen et al. (Scheme 1) [12]. Epoxidation of 7b with MCPBA in CH2 Cl2 followed by addition of acetone and aqueous perchloric acid to the reac-

Fig. 2 – Common bean internode elongation induced by increasing doses of homobrassinolide (2) (open squares ), compound 10a (open cicles ) and compound 12 (open triangles ). The error bars are standard errors.

s t e r o i d s 7 2 ( 2 0 0 7 ) 955–959

In addition, Bs are known to have multiple effects on plant growth and development acting through parallel transduction pathways [15] and to crosstalk to other signals, including auxins, giberellins, abscisic acid and wounding responses [16,17]. A synthetic compound may act on transduction signal cascades with different affinity ratios and as the natural hormone.

4.

Conclusions

We have synthesized and tested the plant growth promoting activity of two new furostanic analogues of brassinosteroids bearing the 5␣-hydroxy-6-oxo moiety. One of the compounds, (22R)-2␣,3␣,5␣-trihydroxy-furostan-6-one (12), produced plant growth stimulation at doses of 2.5 and 5 mg/plants. Though a more detailed interpretation of the above results would require a better understanding of the Bs signal transduction pathways, in our opinion, compound 12 may be useful in the investigation of molecular targets of Bs, and thus its effects on other physiological and biochemical plant responses deserve further research.

Acknowledgements ´ General de Asuntos Financial support was provided by Direccion del Personal Acad´emico (DGAPA-UNAM) via project IN200105. We are indebted to Professor Vladimir Khripach of the Academy of Sciences of Byelorussia for the kind gift of homobrassinolide (2). We are also indebted to Rosa I. del Villar Morales, ´ Georgina Duarte Licsi and Nayeli Lopez Balbiaux (USAI-UNAM) for registering NMR and Mass spectra.

references

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