I. Serbian, et al.
PhytochemistryLetters33(2019)64–69
analysis calcd for C14H16O5 (264.27): C 63.63, H 6.10; found: C 63.35,
H 6.27.
C1‘), 169.8 (C5 + C5‘), 74.1 (C3 + C3‘), 44.9 (C4 + C4‘), 43.4 (C2 +
C2‘), 39.2 (C7 + C7‘), 25.9 (C8 + C8‘) ppm; MS (ESI, MeOH): m/z =
435.1 (100%, [M−H]-), 909.1 (18%, [2M-2H + K]−); analysis calcd
for C16H24N2O12 (436.37): C 44.04, H 5.54, N 6.42; found: C 43.84, H
5.75, N 6.25.
4.4. Dibenzyl (2S, 2'S) 2,2’-((butane-1,4-diylbis(azanediyl))bis(2-
oxoethane-2,1-diyl))bis(2-hydroxypent-4-enoate) (4)
To an ice-cold solution of 3 (1.0 g, 3.8 mmol) in DCM/DMF (1:1;
25 mL), TBTU (1.34 g, 4.18 mmol), HOBt (565 mg, 4.18 mmol) and
DIPEA (2.15 mL, 12.5 mmol) were, and the mixture was stirred for
5 min. 1,4-Diaminobutane (0.15 mL, 1.5 mmol) was added. After stir-
ring for 10 h, the solvent was removed. Usual aqueous work-up fol-
lowed by column chromatography (silica gel, n-hexane/ethyl acetate,
8:1) gave 4 (722 mg, 83%) as a highly viscous, colorless oil; Rf = 0.63
(silica gel, n-hexane/ethyl acetate, 9:1); IR (ATR): 3332w, 2939w,
2870w, 1733s, 1640vs, 1544s, 1499w, 1455m, 1437m, 1411m, 1374m,
1321m, 1213s, 1188vs, 1148s, 1081m, 1029m, 998m, 917m, 738m,
697vs, 660m, 586m cm-1; [α]D = -1.5° (c 0.3, CHCl3); 1H NMR
(500 MHz, CDCl3): δ = 7.38 – 7.30 (m, 10H, HAr), 6.24 (s, NH, 2 H),
5.73 (ddt, J = 17.4, 10.2, 7.3 Hz, 2H, H-5 + H-5‘), 5.25 – 5.16 (m, 4H,
H-8 + H-8‘), 5.12 – 5.01 (m, 4 H, H-6a + H-6b + H6‘-a + H-6‘b), 3.28
– 3.06 (m, 4H, H-13 + H-16), 2.77 (d, J =15.0 Hz, 2H, H-2a + H-2a‘),
2.55 (d, J =15.0 Hz, 2H, H-2b + H-2‘b), 2.46 (qd, J = 13.8, 7.4 Hz, 4H,
H-4 + H-4‘), 1.47 – 1.40 (m, 4H, H-14 + H-15) ppm; 13C NMR
(125 MHz, CDCl3): δ = 174.7 (C-7 + C7‘), 170.4 (C-1 + C-1‘), 135.4
(C-9 + C9‘), 131.4 (C5 + C-5‘), 128.6 (C10 + C10 ‘+ C10‘ ‘+ C10“‘),
128.4 (C11 + C11 ‘+ C11‘ ‘+ C11“ ‘+ C12), 119.6 (C-6), 75.8 (C-3),
67.6 (C-8), 43.8 (C-2 + C-2‘), 43.7 (C-4 + C-4‘), 38.7 (C-13 + C16),
26.5 (C-14+ C-15) MS (ESI, MeOH): m/z = 581.2 (32%, [M+H]+),
603.3 (100%, [M + Na]+); analysis calcd for C32H40N2O8 (580.67): C
66.19, H 6.94, N 4.82; found: C 65.87, H 7.13, N 4.69.
4.7. (3R) 5-Ethoxy-3-(ethoxycarbonyl)-3-hydroxy-5-oxopentanoic acid
(7)
To a suspension of triethylcitrate (10 g, 36.2 mmol) in phosphate
buffer pH = 7.4 (250 mL) Novozym was added (2.5 g), and the mixture
was stirred at 45 °C; the pH was adjusted with 1 M NaOH at 7.4. After 1
equivalent of NaOH has been added, the reaction was allowed to cool to
room temperature. The mixture was extracted with diethyl ether
(2 × 200 mL), acidified with 2 M HCl until pH < 4 and extracted with
ethyl acetate (4 × 200 mL). The combined organic phases were dried
(MgSO4), and the filtrate was concentrated under reduced pressure to
yield 7 (4.03 g, 45%) as a colorless oil; Rf = 0.38 (silica gel, CHCl3/
MeOH, 9:1); [α]D = +5.31° (c 0.35, MeOH); IR (film): ν = 3482s,
2988s, 1740vs, 1374s, 1326s, 1214vs, 1184s, 1128s, 1096s, 1024s cm-1;
1H NMR (400 MHz, DMSO): δ = 4.10 (q, J =7.1 Hz, 2H, H-7), 4.02 (q,
J =7.1 Hz, 2H, H-9), 2.84 (d, J =15.0 Hz, 1H, H-2a), 2.79 (d, J
=15.6 Hz, 1H, H-4a), 2.70 (d, J =15.0 Hz, 1H, H-2b), 2.65 (d, J
=15.5 Hz, 1H, H-4b), 1.19 (t, J =7.1 Hz, 3H, H-8), 1.15 (t, J =7.1 Hz,
3H, H-10) ppm; 13C NMR (100 MHz, DMSO): δ = 172.6 (C-1), 171.0 (C-
5), 169.3 (C-6), 72.9 (C-3), 60.7 (C-7), 60.0 (C-9), 43.0 (C2 + C4), 14.0
(C-8), 13.9 (C-10) ppm; MS (ESI, MeOH): m/z = 248.9 (68%, [M
+H]+), 266.0 (58%, [M + NH4]+), 271.1 (100%, [M + Na]+); ana-
lysis calcd for C10H16O7 (248.23): C 48.39, H 6.50; found: C 48.09, H
6.66.
4.5. (S)-5-[(4-[(S)-4-(Benzyloxy)-3-(carboxymethyl)-3-hydroxy-4-
oxobutanamido)butyl]amino]-3-[(benzyloxy)carbonyl]-3-hydroxy-5-
oxopentanoic acid (5)
4.8. Tetraethyl 2,2'-((((R)-5-ethoxy-5-oxopentane-1,4-diyl)bis
(azanediyl))bis(2-oxoethane-2,1-diyl))(2R,2'R)-bis(2-hydroxysuccinate)
(8)
To a solution of 4 (300 mg, 0.52 mmol) in acetonitrile/water (1:1,
25 mL) at 0 °C ruthenium chloride trihydrate (20 mg, 0.08 mmol) and
sodium metaperiodate (450 mg, 2.1 mmol) were added. The reaction
was allowed to stir for 2 h at 0 °C followed by aqueous workup and
column chromatography (silica gel, 1) CHCl3, 2) MeOH) to yield 5
(210 mg, 63%) as a colorless highly viscous oil; Rf = 0.11 (silica gel, n-
hexane/ethyl acetate, 9:1); 1H NMR (500 MHz, CDCl3): δ = 7.38 – 7.30
(m, 10H, HAr), 5.73 (d, J = 2.1, 4H, H-7 + H-7‘), 3.13 – 3.08 (m, 4H, H-
8 + H-8’), 2.92 (d, J =15.9 Hz, 2H, H-2a + H-2a‘), 2.87 (d, J
=17.0 Hz, 2H, H-2b + H-2‘b), 2.46 (m, 4H, H-4 + H-4‘), 1.46 – 1.42
(m, 4H, H-9 + H-9’) ppm; 13C NMR (125 MHz, CDCl3): δ = 173.5 (C-1
+ C1‘), 171.9 (C-6 + C-6‘), 170.3 (C-5 + C-5’), 135.7 (C-10 + C10‘),
128.1 (C10 + C10 ‘+ C10‘ ‘+ C10“‘), 128.0 (C11 + C11 ‘+ C11‘ ‘+
C11“ ‘+ C12), 73.5 (C-3), 67.1 (C-7), 43.8 (C-2 + C-2‘), 42.8 (C-4 + C-
4‘), 38.5 (C-8 + C8’), 26.50 (C-19+ C-9’) ppm; MS (ESI, MeOH): m/
z = 617.4 (26%, [M+H]+), 639.5 (100%, [M + Na]+); analysis calcd
for C30H36N2O12 (616.61): C 58.44, H 5.88, N 4.54; found: C 58.17, H
6.04, N 4.37.
To a solution of 7 (1.0 g, 3.8 mmol) in DMF (25 mL), TBTU (1.42 g,
4.43 mmol), HOBt (600 mg, 4.43 mmol) and DIPEA (3.05 mL,
17.7 mmol) were added at 0 °C. After 5 min of stirring,
ethylester (256 mg, 1.6 mmol) was added, and stirring wasLc-oornntiitnhuinede
overnight. Usual aqueous work-up followed by chromatography (silica
gel, CHCl3) gave give 8 (772 mg, 88%) as a highly viscous colorless
liquid; Rf = 0.42 (silica gel, CHCl3/ MeOH, 9:1); [α]D= +7.3° (c 0.3,
CHCl3); IR (KBr): ν = 3355br, 2982s, 1732s, 1647s, 1543s, 1370s,
1183m, 1095m, 1024s, 861m cm-1; 1H NMR (500 MHz, CDCl3) δ = 6.96
(s, 1H, NH), 6.63 (s, 1H, NH), 4.51 (m, 1H, H-7), 4.23 (q, J =7.0 Hz, 4H,
H-9 + H-9’), 4.16 (q, J =7.2 Hz, 2H, H-14), 4.11 (q, J = 7.1, 4H, H-11
+ H-11’), 3.25 (m, 2H, H-7’), 2.93 – 2.57 (m, 8H, H-2 + H-2’ + H4 +
H4’), 1.92 – 1.82 (m, 1H, H-8a), 1.70 – 1.60 (m, 1H, H-8b), 1.54 (m, 2H,
H-8’), 1.27 (t, J =7.0 Hz, 6H, H-10 + H-10’), 1.24 (t, J =7.1 Hz, 3H, H-
15), 1.22 (t, J =7.2 Hz, 6H, H-12 + H-12’) ppm; 13C NMR (125 MHz,
DMSO): δ = 173.6 (C-6), 173.5 (C-6’), 171.8 (C-13), 170.1 (C-1), 170.0
(C-1’), 169.6 (C-5), 169.3 (C-5’), 73.7 (C-3 + C3’), 62.3 (C-9 + C-9’),
61.6 (C-14), 61.0 (C-11 + C-11’), 51.9 (C-7), 44.2 (C-4), 44.1 (C-4’),
43.0 (C-2), 42.9 (C-2’), 38.7 (C-7’), 29.4 (C-8), 25.3 (C-8’), 14.0 (C-10
+ C-10’ + C-12 + C-12’ + C-15) ppm; MS (ESI, MeOH): m/z = 621.1
(%, [M+H]+), 643.3 (100%, [M + Na]+); analysis calcd for
C27H44N2O14 (620.65): C 52.25, H 7.15, N 4.51; found: C 51.96, H
7.32, N 4.30.
4.6. (3S, 3’S) Rhizoferrin (6)
Hydrogenation (45 psi) of a solution of 5 (150 mg, 0.26 mmol) in
THF (30 mL) in the presence of 10% Pd/C (20 mg) followed by pur-
ification by column chromatography (C-18, MeOH) gave 6 (105 mg,
91%) as colorless glassy sticky solid; IR (KBr): ν = 3384s, 2922vs,
2852s, 1724vs, 1640vs, 1432vs, 1384vs, 1332s, 1230s, 1120s cm-1; [α]D
= +5.48° (c 0.21, H2O); 1H NMR (500 MHz, D2O): δ = 3.15 - 3.05 (m,
4H, H-7 + H-7‘), 2.94 (d, J =16.0 Hz, 2H, H-2a + H-2‘a), 2.72 (d, J
=16.0 Hz, 2H, H-2b + H-2‘b), 2.63 (d, J =14.1 Hz, H-4a + H-4‘a),
2.58 (d, J =14.5 Hz, 2H, H-4b + H-4‘b), 1.54 - 1.49 (m, 4H, H-8 + H-
8‘) ppm; 13C NMR (125 MHz, D2O): δ = 177.5 (C6 + C6‘), 171.5 (C1 +
4.9. 2-(2-(((S)-1-Carboxy-4-((R)-3,4-dicarboxy-3-hydroxybutanamido)
butyl)amino)-2-oxoethyl)-2-hydroxysuccinic acid (9)
To a suspension of 8 (500 mg, 0.8 mmol) in deionized water
(10 mL), an aqueous solution of Ba(OH)2 (0.2 M, 5 mL) was added. After
stirring for 1 h, 0.5 M sulfuric acid was added until pH = 7. The pre-
cipitated BaSO4 was removed by centrifugation and washed with
67