HETEROCYCLES

Short Paper | Special issue | Vol. 90, No. 2, 2015, pp. 1309-1316
Received, 30th June, 2014, Accepted, 23rd July, 2014, Published online, 5th August, 2014.
DOI: 10.3987/COM-14-S(K)67

■ Synthesis of Rhodotorulic Acid and Its 1,4-Dimethylated Derivative

Michiyasu Nakao, Shintaro Fukayama, Syuji Kitaike, and Shigeki Sano*

Molecular Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokushima, 1-78 Sho-machi, Tokushima 770-8505, Japan

Abstract

Facile syntheses of rhodotorulic acid, isolated from Rhodotorula pilimanae as a siderophore, and its 1,4-dimethylated derivative have been achieved by microwave-assisted cyclization of the corresponding dipeptide precursors.

Siderophores are iron-chelating compounds utilized by bacteria and fungi under iron-limiting conditions.1 Rhodotorulic acid [(S,S)-1] is a dihydroxamate siderophore isolated from Rhodotorula pilimanae,2 and its biological activities3 as well as its iron-chelating ability4 have been investigated. It can be assumed that the diketopiperazine (DKP) ring of (S,S)-1 is biosynthesized starting with L-ornithine, and that two N-hydroxyacetamide moieties serve as a tetradentate ligand for Fe(III) coordination. Therefore, (S,S)-1 has been considered to form a 3 : 2 complex with Fe(III) based on CD spectra and potentiometric titrations, in contrast to hexadentate siderophores such as desferrioxamine B, which forms a 1 : 1 complex with Fe(III).5 The coordination pattern of (S,S)-1 with Fe(III) has also been suggested by electrospray ionization mass spectrometry.6 Despite its interesting structural features, there are only a few examples of (S,S)-1 synthesis.7-10 Herein, we describe a convenient synthesis of (S,S)-1 and its 1,4-dimethylated derivative [(S,S)-2] through microwave-assisted cyclization of the corresponding dipeptide precursors.

Amino acid building blocks, (S)-5-[N-(benzyloxy)acetamido]-2-[(tert-butoxycarbonyl)amino]pentanoic acid [(S)-9] and methyl (S)-2-amino-5-[N-(benzyloxy)acetamido]pentanoate hydrochloride [(S)-10], for dipeptide precursors were synthesized as shown in Scheme 1. Esterification of Boc-L-Glu(OBn)-OH [(S)-3] with methanol using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC•HCl) as a coupling reagent in the presence of a catalytic amount of N,N-dimethyl-4-aminopyridine (DMAP) followed by deprotection of the benzyl ester via catalytic hydrogenolysis under hydrogen with palladium on carbon (10 wt. % loading) afforded the carboxylic acid (S)-5 in 90% yield (2 steps). Formation of a mixed anhydride of (S)-5 with ethyl chloroformate in the presence of N-methylmorpholine (NMM) followed by reduction with sodium borohydride gave the primary alcohol (S)-6 in 89% yield. One-step transformation of the hydroxy group of (S)-6 into the protected hydroxylamino group was performed under Mitsunobu conditions. The reaction of (S)-6 with N-[(2,2,2-trichloroethoxy)carbonyl]-O-benzylhydroxylamine in the presence of diisopropyl azodicarboxylate (DIAD) and triphenylphosphine in THF provided the Mitsunobu product (S)-7. Without further purification, reductive cleavage of the 2,2,2-trichloroethoxycarbonyl (Troc) group of (S)-7 with zinc powder followed by acetylation of the resulting hydroxylamine with acetic anhydride furnished (S)-87,9 in 86% yield (2 steps). Finally, hydrolysis of (S)-8 under aqueous alkaline conditions gave the corresponding N-protected amino acid (S)-9.7,9 In contrast, amino acid ester hydrochloride (S)-109 was obtained by deprotection of the Boc group of (S)-8 under acidic conditions.

Benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent) was used in the condensation of both building blocks (S)-9 and (S)-10 in the presence of N,N-diisopropylethylamine to furnish the dipeptide (S,S)-11 in 95% yield. In previous reports of the synthesis of (S,S)-1, two steps have been required to construct the DKP structure via N-terminal deprotection and intramolecular cyclization of the dipeptide precursor.7-10 In addition, a long reaction time has been needed for intramolecular cyclization. On the other hand, microwave irradiation has been reported to be efficient for a one-pot conversion of N-Boc-dipeptide methyl esters into DKPs with a short reaction time.11 We therefore attempted a one-pot conversion of (S,S)-11 into DKP (S,S)-12 using microwave irradiation. As a result, removal of the Boc group followed by intramolecular cyclization of (S,S)-11 under microwave irradiation with a single-mode microwave reactor (InitiatorTM 60; Biotage AB) at 170 °C in a mixed solvent of water/methanol for 10 min furnished the DKP (S,S)-12 in 63% yield. Finally, catalytic hydrogenolysis of (S,S)-12 under hydrogen with palladium on carbon (10 wt. % loading) provided rhodotorulic acid [(S,S)-1] in 80% yield. Furthermore, N-methylation of the DKP ring of (S,S)-12 followed by catalytic hydrogenolysis of the resultant (S,S)-13 afforded 1,4-dimethylated rhodotorulic acid [(S,S)-2]. The structures of (S,S)-1 and (S,S)-2 were confirmed by spectroscopic methods. In general, DKP derivatives have poor solubility in various solvents due to their intermolecular hydrogen bonding through the amide moiety of the DKP ring.12 Therefore, only a few solvents, including water and dimethylsulfoxide (DMSO), have been found to be capable of dissolving (S,S)-1. However, (S,S)-2 was found to be soluble in water, DMSO, methanol, ethanol, chloroform, and ethyl acetate. This enhanced solubility is likely due to the disappearance of intermolecular hydrogen bonding as a result of N-methylation.
In conclusion, we have presented the synthesis of rhodotorulic acid [(S,S)-1] and its 1,4-dimethylated derivative [(S,S)-2] using a microwave-assisted cyclization of the corresponding common dipeptide precursor (S,S)-11 as a key step. Intriguingly, (S,S)-2 was found to be more soluble in various organic solvents than (S,S)-1. Derivatization of (S,S)-1 and (S,S)-2 toward the synthesis of novel iron-chelating compounds is currently underway and will be reported in due course.

EXPERIMENTAL
All melting points were determined on a Yanagimoto micro melting point apparatus and are uncorrected. IR spectra were obtained using a JASCO FT/IR-6200 IR Fourier transform spectrometer. 1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were recorded on a Bruker AV500 spectrometer. Chemical shifts are given in δ values (parts per million) using tetramethylsilane (TMS) as an internal standard. Electron spray ionization mass spectra (ESIMS) were recorded on a Waters LCT Premier spectrometer. Elemental combustion analyses were performed using a J-SCIENCE LAB JM10. The microwave-assisted reaction was performed utilizing an automated single-mode microwave synthesizer (InitiatorTM 60; Biotage AB). All reactions were monitored by TLC employing 0.25-mm silica gel plates (Merck 5715; 60 F254). Column chromatography was carried out on silica gel [Kanto Chemical 60N (spherical, neutral); 63-210 mm] or [Fuji Silysia Chemical PSQ 60B (spherical)]. Anhydrous THF, CH2Cl2, and DMF were used as purchased from Kanto Chemical. N-Methylmorpholine (NMM) and N,N-diisopropylethylamine were distilled prior to use. All other reagents were used as purchased.

Methyl (S)-5-[N-(Benzyloxy)acetamido]-2-{(S)-5-[N-(benzyloxy)acetamido]-2-[(tert-butoxycarbonyl)amino]pentanamide}pentanoate [(S,S)-11]
To a solution of (S)-9 (618 mg, 1.62 mmol) and (S)-10 (537 mg, 1.62 mmol) in anhydrous CH2Cl2 (6 mL) were added BOP reagent (1.1 g, 2.44 mmol) and N,N-diisopropylethylamine (552 µL, 3.25 mmol) at 0 °C under argon. The reaction mixture was allowed to warm to rt and stirred for 24 h. The reaction mixture was treated with 5% citric acid aq (2 mL) and then extracted with CHCl3 (50 mL x 3). The extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The oily residue was purified by silica gel column chromatography [Silica Gel PSQ 60B: CHCl3–MeOH (100:0 to 10:1)] to afford (S,S)-11 (1.0 g, 95%). Colorless oil; [α]D19 +5.6 (c 0.51, CHCl3); 1H NMR (500 MHz, CDCl3) δ 1.43 (s, 9H), 1.47–1.88 (m, 8H), 2.09 (s, 3H), 2.11 (s, 3H), 3.41–3.53 (m, 1H), 3.60–3.73 (m, 2H), 3.66 (s, 3H), 4.12–4.27 (m, 1H), 4.32–4.42 (m, 1H), 4.48–4.56 (m, 1H), 4.76–4.88 (m, 4H), 5.22–5.28 (m, 1H), 7.08–7.17 (m, 1H), 7.33–7.42 (m, 10H); 13C NMR (125 MHz, CDCl3) δ 20.4, 20.5, 23.0, 23.2, 28.3, 29.1, 30.6, 43.6, 44.7, 51.9, 52.2, 52.3, 76.31, 76.34, 79.6, 128.72, 128.74, 128.96, 129.01, 129.19, 129.24, 134.3, 134.4, 155.8, 172.37, 172.45 (two overlapping singlets), 173.16; 13C NMR (125 MHz, C6D6) δ 20.5 (two overlapping singlets), 23.4, 23.7, 28.4, 29.2, 31.1, 43.9, 45.2, 51.7, 52.2, 52.7, 76.1, 76.3, 79.1, 128.77, 128.79, 128.8, 128.9, 129.4, 129.5, 135.1, 135.4, 156.3, 172.1, 172.8, 172.9, 173.1; IR (neat) 3304, 2978, 2935, 1743, 1659, 1499 cm-1; ESI-MS m/z: calcd for C34H48N4NaO9 [M+Na]+, 679.3319; found, 679.3350.

N,N’-{[(2S,5S)-3,6-Dioxopiperazine-2,5-diyl]bis(propane-3,1-diyl)}bis[N-(benzyloxy)acetamide] [(S,S)-12]
A suspension of (S,S)-11 (611 mg, 0.931 mmol) in a mixed solvent of H2O (15 mL) with MeOH (5 mL) was irradiated at 170 °C for 10 min utilizing a Biotage Initiator® microwave synthesizer. The reaction mixture was treated with H2O (20 mL) and then extracted with AcOEt (50 mL x 3). The extract was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The oily residue was purified by silica gel column chromatography [Silica Gel 60N: CHCl3–MeOH (98:2 to 85:15)] to afford (S,S)-12 (305 mg, 63%). Colorless powder (MeOH–Et2O); mp 149–150 °C (lit.7 127–129 °C, lit. 9 129–131 °C, and lit.10 97–99 °C); [α]D27 -20.5 (c 1.03, MeOH) {lit.7 [α]D25 -16.5 (c 0.67, MeOH), lit.9 [α]D13 -16.4 (c 1.01, EtOH), and lit.10 [α]D20 -16.4 (c 1.01, EtOH)}; 1H NMR (500 MHz, CD3OD) δ 1.67–1.85 (m, 8H), 2.03 (s, 6H), 3.63–3.73 (m, 4H), 3.98 (t, J = 5.2 Hz, 2H), 4.87 (s, 4H), 7.34–7.43 (m, 10H); 13C NMR (125 MHz, CD3OD) δ 20.5, 23.6, 32.4, 45.6, 55.7, 77.2, 129.8, 130.0, 130.7, 136.1, 170.2, 174.5; IR (KBr) 3193, 3043, 2953, 2886, 1665, 1455 cm-1; ESI-MS m/z: calcd for C28H36N4NaO6 [M+Na]+, 547.2533; found, 547.2525. Anal. Calcd for C28H36N4O6: C, 64.10; H, 6.92; N, 10.68. Found: C, 63.96; H, 6.91; N, 10.53%.

N,N’-{[(2S,5S)-3,6-Dioxopiperazine-2,5-diyl]bis(propane-3,1-diyl)}bis(N-hydroxyacetamide) [Rhodotorulic Acid, (S,S)-1]
The mixture of (S,S)-12 (100 mg, 0.191 mmol) and 10% Pd–C (20 mg, 0.019 mmol) in MeOH (3 mL) was stirred at rt for 1 h under hydrogen. The reaction mixture was filtered and concentrated in vacuo to afford (S,S)-1 (53 mg, 80%). Colorless powder (H2O); mp >217 °C (dec) [lit.7 217–218 °C, lit.8 229–232 °C, lit.9 216–218 °C (dec), and lit.10 217–218.5 °C (dec)]; [α]D27 -30.2 (c 0.16, H2O) {lit.8 [α]D -30.5 (c 0.67, AcOH), lit.9 [α]D17 -30.4 (c 0.5, H2O), lit.10 [α]D25 -28.8 (c 1.00, H2O)}; 1H NMR (500 MHz, DMSO-d6) δ 1.50–1.72 (m, 8H), 1.97 (s, 6H), 3.43–3.52 (m, 4H), 3.79–3.86 (m, 2H), 8.16 (brs, 2H), 9.72 (brs, 2H); 13C NMR (125 MHz, DMSO-d6) δ 20.2, 22.0, 30.2, 46.7, 53.7, 167.7, 170.0; IR (KBr) 3187, 3086, 2867, 1686, 1594, 1517, 1473, 1448 cm-1; ESI-MS m/z: calcd for C14H24N4NaO6 [M+Na]+, 367.1594; found, 367.1588. Anal. Calcd for C14H24N4O6: C, 48.83; H, 7.02; N, 16.27. Found: C, 48.53; H, 6.99; N, 16.14%.

N,N’-{[(2S,5S)-1,4-Dimethyl-3,6-dioxopiperazine-2,5-diyl]bis(propane-3,1-diyl)}bis[N-(benzyloxy)acetamide] [(S,S)-13]
NaH (50–72%, 13.6 mg, 0.284 mmol) was added to a solution of (S,S)-12 (49.7 mg, 0.0947 mmol) in anhydrous DMF (2 mL) and stirred at 0 °C for 5 min under argon. After adding MeI (17.7 µL, 0.284 mmol), the mixture was stirred at 0 °C for 30 min under argon. The reaction mixture was treated with 1N HCl aq (1 mL) and then extracted with AcOEt (20 mL x 3). The extract was washed with sat. Na2S2O3 aq (5 mL) and H2O (5 mL x 3). The organic layer was dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The oily residue was purified by silica gel column chromatography [Silica Gel PSQ 60B: CHCl3–MeOH (20:1 to 10:1)] to afford (S,S)-13 (43.2 mg, 83%). Colorless oil; [α]D20 +11.4 (c 0.90, CHCl3); 1H NMR (500 MHz, CDCl3) δ 1.60–1.92 (m, 8H), 2.09 (s, 6H), 2.89 (s, 6H), 3.58–3.68 (m, 2H), 3.74–3.87 (m, 4H), 4.81 (dd, J = 10.5, 13.7 Hz, 4H), 7.34–7.43 (m, 10H); 13C NMR (125 MHz, CDCl3) δ 20.5, 23.5, 30.5, 32.6, 44.1, 61.7, 76.4, 128.8, 129.1, 129.2, 134.3, 165.7, 172.3; IR (neat) 2937, 2878, 1660, 1454, 1403 cm-1; ESI-MS m/z: calcd for C30H40N4NaO6 [M+Na]+, 575.2846; found, 575.2815.

N,N’-{[(2S,5S)-1,4-Dimethyl-3,6-dioxopiperazine-2,5-diyl]bis(propane-3,1-diyl)}bis(N-hydroxyacet-amide) [1,4-Dimethylated Rhodotorulic Acid, (S,S)-2]
The mixture of (S,S)-13 (24.2 mg, 0.0438 mmol) and 10% Pd–C (2.3 mg, 0.00219 mmol) in MeOH (1 mL) was stirred at rt for 2 h under hydrogen. The reaction mixture was filtered and concentrated in vacuo to afford (S,S)-2 (12 mg, 80%). Colorless prism (CHCl3–Et2O); mp 134–135.5 °C; [α]D28 +31.0 (c 0.45, CHCl3); 1H NMR (500 MHz, CDCl3) δ 1.67–1.89 (m, 6H), 2.05–2.18 (m, 2H), 2.16 (s, 6H), 2.98 (s, 6H), 3.57–3.67 (m, 2H), 3.77–3.92 (m, 4H), 9.35 (brs, 2H); 13C NMR (125 MHz, CDCl3) δ 20.6, 22.2, 30.9, 32.9, 47.3, 61.9, 166.7, 172.7; IR (KBr) 3351, 3115, 2939, 2868, 1663, 1636, 1600 cm-1; ESI-MS m/z: calcd for C16H28N4NaO6 [M+Na]+, 395.1907; found, 395.1920. Anal. Calcd for C16H28N4O6: C, 51.60; H, 7.58; N, 15.04. Found: C, 51.30; H, 7.51; N, 14.91%.

ACKNOWLEDGEMENTS
This work was supported in part by a Grant for the Regional Innovation Cluster Program (Global Type) promoted by MEXT.

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