Nandrolone Decanoate Transformation by Neurospora crassa.

Abstract

Neurospora crassa. Shear & B.O. Dodge (Sordariaceae), the red bread mold, when incubated with exogenous nandrolone decanoate for 6 days at 25°C, produced four steroid metabolites. The microbial products were purified by preparative thin-layer chromatography and high-performance liquid chromatography followed by their identification through ¹H, ¹³C NMR and other spectroscopic data. The fermentation yielded estr-4-en-3,17-dione (II), 17β.-hydroxyestr-4-en-3-one (III), 6β.,17β.-dihydroxyestr-4-en-3-one (IV), and 9β.,17β.-dihydroxyestr-4-en-3-one (V). Characteristics observed were ester hydrolysis, hydroxyl group oxidation, and 9α.- or 6β.-hydroxylations.

Keywords:

• Microbial transformation
• nandrolone decanoate
• Neurospora crassa
• steroid

Introduction

The importance of microbial transformation of steroids was realized for the first time in 1952 when Murray and Peterson of Upjohn Co. patented the process of 11α.-hydroxylation by a Rhizopus. species. Then, many other microorganisms were found as the biocatalysts of steroid transformations, and several reactions such as α.- or β.-hydroxylation, double bond reduction, oxidation, and so forth, have been carried out on steroid compound modifications in pharmaceutical industries (Smith, 1984).

Among the filamentous fungi, strains belonging to the genera of Aspergillus. and Rhizopus. have been widely used for transformation of steroids to produce pharmaceutically active compounds (Mahato & Makherjee, 1984; Mahato & Banerjee, 1985; Mahato et al., 1989; Mahato & Mazudmer, 1993; Mahato & Garai, 1997). Neurospora crassa. Shear & B.O. Dodge (Sordariaceae) has also been applied for transformation of some steroid substances. Incubation of desoxycorticosterone with N. crassa. to a hydroxylated derivative was reported by Stone in 1955 (Stone et al., 1955). Progesterone was also considered as a steroid substance in transformation process by N. crassa. (Charney & Herzog, 1967). In 1973, Maugras et al. reported the reduction of ketone function at C-17 to a 17-hydroxyl group and hydroxylation at C-12 to 12β.-form by this fungus (Maugras et al., 1973a). They also showed the transformation of estradiol to estrone and strone to estradiol in the culture of this fungus in another study (Maugras et al., 1973b).

Neurospora crassa. has been highly characterized genetically and biochemically (Perkins et al., 1982; Mishra, 1991). The fungus has an efficient and well-established DNA transformation system. It is also of the fastest growing of all filamentous fungi (doubling time under optimal condition 2 h) and produces no toxic secondary metabolite (Fincham, 1989). In this study, we examined Neurospora crassa. for transformation of nandrolone decanoate.

Materials and Methods

Chemicals

Nandrolone decanoate was kindly donated by Iran Hormone Pharmaceutical Co. (Tehran, Iran), which had been purchased from Gedeonrichter (Budapest, Hungary). Sabouraud dextrose agar and broth were purchased from Merck (Darmstadt, Germany). All other reagents and solvents were of analytical grade.

Instruments

Melting points (mp) were determined on a Reichert-Jung hot-stage melting-point apparatus and are uncorrected. Optical rotations were measured in 1-dm cells on a Perkin-Elmer 142 automatic spectropolarimeter. ¹H and ¹³C NMR spectra were recorded using FTNMR Varian Unity plus spectrometer at 400 and 100 MHz, respectively, in CDCl₃ with tetramethylsilane (TMS) as internal standard. Chemical shifts (δ) are given in parts per million (ppm) relative to TMS. Coupling constants (J.) are given in Hertz (Hz). Infrared (IR) spectra were recorded on a Magna-IR 550 Nicolet FTIR spectrometer. Mass spectra (MS) were obtained with a Finnigan MAT TSQ-70 instrument by electron impact (EI) at 70 eV. Thin-layer chromatography (TLC) and preparative TLC were performed, respectively, on 0.25- and 0.5-mm-thick layers of silica gel G (Kieselgel 60 HF_254+366, Merck). Layers were prepared on glass plates and activated at 105°C 1 h before use. Chromatography was performed with acetone/hexane (1:1) and visualized by spraying the plates with a mixture of methanol/sulfuricacid (6:1) and heating in an oven at 100°C for 3 min until the colors developed. The compounds were also visualized under UV lamp (Strstedt-Gruppe HP-UVIS) at 254 nm.

High-performance liquid chromatography (HPLC) was performed on a Waters HPLC system (Waters, Milford, MA, USA), which consisted of a Waters model 600 pump and a Waters 486 UV/VIS detector adjusted to 330 nm connected to 746 Data Module Integrator. Samples were injected to a 7725 Rheodyne injector system with a 20 µl sample loop. Separation was carried out using a C18 Novapak reversed-phase column (5 µm, 150 × 3.9 mm).

Microorganism

The strain of Neurospora crassa. FGSC 4335 was obtained from the Fungal Genetics Stock Center, University of Kansas. It was maintained on Sabouraud-4%-dextrose agar slope and freshly subcultured before using in transformation experiment.

Incubation conditions

Ten 500 ml Erlenmeyer flasks, each containing 100 ml of liquid medium of Sabouraud-2%-dextrose broth, were inoculated with freshly obtained spores from agar slant cultures and incubated for 12 h at 25°C in a rotary shaker (150 rpm). The spores were collected with a sterile normal saline solution containing 0.1% Tween 80. Nandrolone decanoate (1 g) was dissolved in 20 ml of absolute ethanol. Two ml of the ethanol solution was added to each 500 ml Erlenmeyer flask and incubation continued for 6 days at the same conditions. The control was similarly processed without the microorganism.

Biotransformation, product isolation, and purification

At the end of the fermentation period, media were extracted with chloroform, and the extract was washed with water, dried over sodium sulfate, and evaporated under reduced pressure. To analyze the results of bioconversion, TLC was used with acetone/hexane (1:1), and the metabolites were visualized under UV lamp at 254 nm. Preparative TLC was used with silica gel G on glass plates 20 × 20 and with thickness of 0.5 mm to purify the metabolites, and they were separated using the above-mentioned solvent system. All metabolites were crystallized from methanol. In order to find the purity as well as obtaining the biotransformation yield of each metabolite, HPLC was applied. Twenty µl of concentrated extract was injected to a C18 reverse-phase column. Elution was done by isocratic method using methanol/water (45:55) with a flow rate of 1 ml min⁻¹. Detection was done by UV at 254 nm.

Results

The crude extract obtained from 6 days incubation of N. crassa. in the presence of nandrolone decanoate resulted in four main products (II–V), in addition to the substrate (I) (Fig. 1) as follows.

Figure 1 The structures of substrate and biotransformed metabolites. (I) Nandolone decanoate, (II) estr-4-en-3,17-dione, (III) 17β.-hydroxyestr-4-en-3-one, (IV) 6β.,17β.-dihydroxyestr-4-en-3-one, (V) 9α.,17β.-dihydroxyestr-4-en-3-one.

Estr-4-en-3,17-dione (II).

m.p. 168–170°C lit (Rosenberger et al., 1978) 168–171°C; [α]_D + 138 (CHCl₃) lit (Rosenberger et al., 1978) [α]_D + 139.2; IR νmax 2934, 1706, 1662, 1615 cm⁻¹; MS (EI) m/z. (%) 272 (100) (M⁺, C₁₈H₂₄O₂), 243 (11), 228 (23), 186 (38), 149 (16), 110 (34), 97 (13), 85 (13), 83 (19); ¹H NMR (CDCl₃, DMSO) δ 0.92 (3H, s, H-18), 5.86 (1H, s, H-4); R_f in acetone/hexane (1:1):0.63. Two carbonyl groups are clear according to the IR and ¹³C NMR data (see Table 1) in addition to the molecular ion at m/z. 272. It was identified as estr-4-en-3,17-dione, which had been reported (Hanson et al., 1996).

Table 1.. ¹³C NMR signals of metabolites (δ in ppm downfield from TMS, in CDCl₃)

	Compounds
Carbon atom	I	II	III	IV	V
1	27.4	25.7	26.0	25.9	20.6
2	36.4	36.5	36.3	36.3	36.2
3	199.7	199.6	199.9	199.8	199.7
4	124.5	124.9	124.4	125.4	127.1
5	166.2	165.6	166.8	168.0	164.1
6	35.3	35.2	35.4	71.8	34.6
7	31.7	31.3	30.6	38.1	24.7
8	40.1	39.9	40.4	33.6	43.3
9	49.4	49.5	49.6	49.5	74.2
10	42.4	42.5	42.5	37.7	47.6
11	26.5	26.6	26.5	26.1	31.9
12	36.5	29.9	36.4	36.4	32.2
13	42.6	47.6	42.8	43.2	42.8
14	49.3	50.1	49.5	49.7	43.6
15	23.2	21.6	23.1	23.1	23.0
16	30.5	35.7	30.2	30.4	30.3
17	82.1	220.3	81.5	81.7	81.3
18	12.0	13.8	11.0	11.1	10.2

(I) Nandolone decanoate, (II) estr-4-en-3,17-dione, (III) 17β.-hydroxyestr-4-en-3-one, (IV) 6β.,17β.-dihydroxyestr-4-en-3-one, (V) 9α.,17β.-dihydroxyestr-4-en-3-one.

17β-Hydroxyestr-4-en-3-one(III)

m.p. 112–115°C lit (Wilds & Nelson, 1953) m.p. 111–112°C and 123–124°C (dimorphic crystals); [α]_D + 55 (CHCl₃), lit (Wilds & Nelson, 1953) [α]_D + 55; IR νmax 3513, 2929, 1675, 1630 cm⁻¹ MS (EI) m/z. (%) 274 (100) (M⁺, C₁₈H₂₆O₂), 256 (26), 216 (29), 201 (65), 173 (21), 147 (33), 110 (59), 85 (52); ¹H NMR (CDCl₃, DMSO) δ 0.80 (3H, s, H-18), 3.66 (1H, t, J. = 8.8 Hz, H-17), 5.83 (1H, s, H-4); R_f in acetone/hexane (1:1): 0.46. This compound had been also reported by Hanson et al. (1996).

6β,17β-Dihydroxyestr-4-en-3-one. (IV)

m.p. 208–211°C, lit (Hanson et al., 1996) 212–215°C; [α]_D − 50 (CHCl₃), lit (Hill et al., 1991) [α]_D − 53, IR νmax 3427, 2913, 1660, 1625 cm⁻¹; MS (EI) m./z. (%) 290 (7) (M⁺, C₁₈H₂₆O₃), 272 (43), 256 (15), 201 (27), 159 (60), 149 (100), 146 (20), 83 (24), 71 (39), 57 (26); ¹H NMR (CDCl₃, DMSO) δ 0.81 (3H, s, H-18), 3.67 (1H, t, J. = 8.4 Hz, H-17), 4.38 (1H, t, J. = 2.8 Hz, H-6), 5.89 (1H, s, H-4); R_f in acetone/hexane (1:1):0.18. The peak at δ 4.38 in ¹H NMR spectra clearly showed the existance of another hydroxyl group in addition to 17β.-hydroxy on nandrolone. This data was suppoted by ¹³C NMR spectra. The signal at δ 71.8 in ¹³C NMR spectra was assigned C-6 where the hydroxyl group caused downfield shift of C-6 resonance. The chemical shifts of other carbons in 6β.,17β.-dihydroxyestr-4-en-3-one were similar to that of literature report (Hanson et al., 1996).

9α,17β-Dihydroxyestr-4-en-3-one. (V)

m.p. 190–193°C, lit (Farkas & Owen, 1966) 191–193°C; [α]_D + 14.5 (CHCl₃); IR νmax 3456, 2918, 1655, 1632 cm⁻¹; MS (EI) m./z. (%) 290 (16) (M⁺, C₁₈H₂₆O₃), 272 (8), 256 (10), 199 (10), 154 (23), 123 (32), 110 (100), 90 (13); ¹H NMR (CDCl₃, DMSO) δ 0.81 (3H, s, H-18), 2.28 (2H, m, H-6), 2.49 (1H, dd, J. = 11 Hz, 4.6 Hz, H-10), 3.72 (1H, t, J. = 8.4, H-17), 5.95 (1H, s, H-4); R_f in acetone/hexane (1:1):0.13. The chemical shift at δ 74.2 in ¹³C NMR showed the addition of another hydroxyl group on nandrolone, but no downfield proton resonance was observed in ¹H NMR spectra around δ 3.5–4.5 excluding the peak at δ 3.72 which belonged to C-17. These data confirmed that the second hydroxyl group in compound V should be attached to C-8, C-9, C-10, or C-14 where no α.-proton was connected to C–OH group. A proton as dd at δ 2.49 for H-10 concluded that the hydroxyl group should be at C-9, as this proton appeared at δ 2.46 in nandrolone (III) as oct.

The HPLC profile is shown in Figure 2. An isocratic elution of these compounds using methanol/water (45:55) as mobile phase in HPLC separation resulted in a good resolution of the metabolites and unconverted substrate. The retention times of four bioconverted metabolites were 4.97, 7.51, 21.45, and 34.37 min for compounds IV, V, II, and III, respectively. In this method, the retention time of nandrolone decanoate (I) was 121.93 min. The yields of products expressed as percentage of sum of total transformed products and the remaining substrate by direct computational integration of the individual peaks were 1.83%, 22.3%, 3.2%, and 5.5% for the metabolites II, III, IV, and V, respectively. The percentage of unconverted substrate was 57.9%. The HPLC analysis revealed that few other metabolites were also found in the transformed mixture, which were not purified for characterization, due to trace quantities.

Figure 2 HPLC chromatogram of 6-day nandrolone decanoate transformation by N. crassa.: nandolone decanoate (I) (R_t = 121.93 min), estr-4-en-3,17-dione (II) (R_t = 21.45 min), 17β.-hydroxyestr-4-en-3-one (III) (R_t = 34.37 min), 6β.,17β.-dihydroxyestr-4-en-3-one (IV) (R_t = 6.14 min), and 9α.,17β.-dihydroxyestr-4-en-3-one (V) (R_t = 7.51 min).

Daily sampling of the fermentation broth was also performed every 24 h. Samples were extracted and analyzed using HPLC method (Fig. 3). According to the curves in Fig. 3, transformation of the substrate started at the first day, and all the metabolites were detectable in the third day. The compounds II, IV, and V were not increased after 6 days incubation, whereas the enzymatic ester hydrolysis of nandrolone decanoate into nandrolone was still continued after 6 days (see Fig. 3).

Figure 3 Transformation of nandrolone decanoate with N. crassa. in 7 days incubation resulted in compounds II (▪), III (♦), IV (▴), and V (•).

Discussion

The results indicated the ability of Neurospora crassa. in bioconversion of nandrolone decanoate. The biotransformation characteristics (Fig. 1) were ester hydrolysis, hydroxyl group oxidation, and 9α.- or 6β.-hydroxylations of the substrate (I).

Nandrolone has been used in many studies as a substrate of biotransformation procedures. Huszcza et al. (2003) isolated 10β-hydroxy-19-norandrost-4-en-3,17-dione from the culture of Botrytis cinerea. containing nandrolone (Huszcza & Dmochowska-Gladysz, 2003). 10β.-Hydroxylated derivatives of nandrolone were also reported by Favero and Lin by Rhizopus arrhizus. and Curvularia lunata., respectively (Lin et al., 1969; Favero et al., 1977). It seems that 10β.-hydroxylation is a common bioreaction on nandrolone by other fungi; however, no 10β.-hydroxylated derivative was observed in this study.

There are also some reports on hydroxylation of nandrolone at C-11 (Lin et al., 1969; Petzoldt et al., 1984), C-12 (Lin et al., 1969), C-14 (Lin et al., 1969), C-15 (Yoshihama et al., 1989), and C-16 (Holland et al., 1989), but such metabolites were not detected in the current work.

This study showed C-6 or C-9 as two suitable sites for hydroxylation of nandrolone with N. crassa.. Although 6β.-hydroxylation has been previously reported in microbiological transformation of nandrolone by some fungi (Wilds & Nelson, 1953; Favero et al., 1977), no literature report was found on 9α.-hydroxylation of this substrate. Because steroid 9α.-hydroxylation is useful for production of some pharmaceutically interesting steroids such as dexamethazone, betamethasone, fluocinolone, and so forth (Kieslich, 1980; Smith, 1984), the strain of N. crassa. we applied can be considered as a suitable biocatalyst for this purpose.

Acknowledgments

The authors would like to acknowledge Mr. Khosro M. Abdi and Mr. Majid Darabi for their kind collaborations in spectral studies and Dr. Gholamreza Zarrini for his valuable advice on fungal culture.