M.K. Pangopoulos, et al.
Bioorganic Chemistry 96 (2020) 103653
portionwise added to the solution of 6. After 30 min (TLC,
hexane:EtOAc 80:20) showed complete conversion of 6. Then HCl
2H), 1.64 (quint, J = 7.1 Hz, 2H), 1.35 (s, 1H), 0.97 (t, J = 7.5 Hz, 3H);
1
3
C NMR (100 MHz, CDCl ): δ 132.33 (CH), 129.70 (CH), 128.84 (CH),
3
(
aqueous, 1.4 M, 51 mL) was dropwise added and the product was
128.76 (CH), 128.55 (CH), 128.53 (CH), 128.40 (CH), 128.37 (CH),
extracted with hexane:Et
phases washed with brine (2 × 50 mL) and dried (MgSO
was purified by column chromatography (SiO , hexane:EtOAc 90:10).
2
O (2:1, 3 × 50 mL), the combined organic
128.15 (CH), 127.29 (CH), 62.78 (CH
25.90 (CH ), 25.87 (CH ), 25.81 (CH
14.53 (CH
mass calc for C19H30O 274.2297, found: 274.2295.
2
), 32.76 (CH
2
), 25.91 (CH
2
2
),
4
). The product
2
3
2
2
), 23.85 (CH
2
), 20.82 (CH
),
−
1
2
); IR: 3338 (broad), 3012, 2963, 2932 cm ; HRMS: Exact
After removal of solvent the alcohol 7 was obtained as a colorless oil
(
2.56 g, 75%). The spectral data were in accord with those earlier re-
1
ported [21]. H NMR (400 MHz, CDCl
3
): δ 5.57 (m, 1H), 5.47–5.31 (m,
9
2
H), 3.68 (t, J = 6.4 Hz, 2H), 2.90–2.83 (m, 8H), 2.39 (q, J = 6.8 Hz,
4.5. 2-(((4Z,7Z,10Z,13Z,16Z)-nonadeca-4,7,10,13,16-pentaen-1-yl)oxy)
acetic acid (3-oxa n-3 DPA, 5)
H), 2.10 (quint, J = 7.2 Hz, 2H), 1.46 (s, 1H), 1.00 (t, J = 7.5 Hz, 3H);
1
3
C NMR (100 MHz, CDCl
28.34 (CH), 128.31 (CH), 128.04 (CH), 127.99 (CH), 127.87 (CH),
27.02 (CH), 125.66 (CH), 62.22 (CH ), 30.83 (CH ), 25.77 (CH ),
5.66 (CH ), 25.64 (CH ), 25.55 (CH ), 20.57 (CH ), 14.28 (CH ).
3
): δ 132.06 (CH), 131.14 (CH), 128.60 (CH),
1
1
2
The alcohol 9 (0.21 g, 0.77 mmol) in toluene (10 mL) was cooled to
2
2
2
0 °C. NaOH (50% aqueous solution, 10 mL) was added, followed by
2
2
2
2
3
addition of n-Bu
4
NHSO (0.026 g, 0.077 mmol). The mixture was left
4
stirring for one hour at 0 °C. tert-Butyl bromoacetate (0.17 mL,
4.3. 4Z,7Z,10Z,13Z,16Z)-nonadeca-4,7,10,13,16-pentaenenitrile (8)
1.2 mmol) was added dropwise and the reaction mixture was left stir-
ring overnight at room temperature. Hexane:H O (1:1, 40 mL) was
2
Alcohol 7 (2.56 g, 9.80 mmol) was dissolved in dry CH Cl followed
2
2
added and the phases were separated. The aqueous phase was extracted
by the addition of Et
3
N (2.75 mL, 19.7 mmol). The solution was cooled
with Et
2
O (4 × 20 mL), dried (Na
2
SO ) and concentrated under re-
4
to 0 °C. Methanesulfonyl chloride (1.52 mL, 19.7 mmol) was added
dropwise and the reaction mixture was left stirring for 10 min at this
temperature. The solution was allowed to warm to room temperature
and stirred at this temperature for three hours when brine (20 mL) was
added dropwise. The resulting mixture was extracted with EtOAc
duced pressure. This gave tert-butyl ester 11 (0.34 g, 0.87 mmol) that
was used directly in the next reaction. The ester was dissolved in
THF:MeOH:H
2
O (2:2:1) (43 mL) and cooled to 0 °C. LiOH·H O (1.3 g,
2
30 mmol) was added. All the starting material was consumed after three
hours, and the mixture was allowed to heat to room temperature. HCl
(
3 × 30 mL) and the combined organic extracts were washed with a
(37%) was added until pH 2. The solution was diluted with CH
2
2
Cl , and
saturated NaHCO
3
-solution (2 × 20 mL) and brine (2 × 10 mL), dried
) and concentrated under reduced pressure to give crude 8
3.13 g) as a brown oil, which was used directly in the next reaction.
the organic phase was separated. The aqueous layer was extracted with
CH Cl (4 × 20 mL), dried (Na SO ) and concentrated under reduced
pressure. The resulting oil was purified by column chromatography
(
(
Na
2
SO
4
2
2
2
4
The crude mesylate of 7 was dissolved in DMSO (25 mL) and KCN
(SiO
2
, 100% CH
2
Cl
2
, followed by CH
2
Cl :MeOH 97.5:2.5), giving the
2
(
0.90 g, 13.9 mmol) was added. The mixture was heated to 70 °C for
two hours. Water (90 mL) was added and the mixture was extracted
with EtOAc (3 × 50 mL), dried (Na SO ) and concentrated under re-
pure target molecule (0.16 g, 64% yield over two steps) as a yellow oil.
1
H NMR (400 MHz, CDCl ): δ 5.43–5-30 (m, 10H), 4.10 (s, 2H), 3.57 (t,
3
2
4
J = 6.6 Hz, 2H), 2.88–2.78 (m, 8H), 2.15 (q, J = 6.4 Hz, 2H), 2.07
(quintet, J = 7.4 Hz, 2H), 1.71 (quintet, J = 7.2 Hz, 2H), 0.98 (t,
duced pressure. The resulting crude oil was purified by column chro-
1
3
matography (SiO
2
; hexane:EtOAc 15:1) to afford nitrile 8 (1.7 g,
J = 7.5 Hz, 3H); C NMR (600 MHz, CD OD): δ 174.18 (CO), 132.78
3
1
6
.2 mmol; 63% over two steps) as a pale yellow oil. H NMR (400 MHz,
(CH), 130.33 (CH), 129.48 (CH), 129.43 (CH), 129.32 (CH), 129.14
(CH), 129.13 (CH), 129.00 (CH), 128.92 (CH), 128.18 (CH), 71.97
CDCl
3
): δ 5.54 (m, 1H), 5.45–5.28 (m, 9H), 2.86–2.80 (m, 8H), 2.38 (m,
1
3
4
H), 2.08 (quint, J = 7.3 Hz, 2H), 0.97 (t, J = 7.5 Hz, 3H); C NMR
(CH
(CH
2
2
), 68.81 (CH
), 26.44 (CH
2
2
), 30.52 (CH
2
), 26.57 (CH
2
), 26.55 (CH
2
), 26.51
); IR:
(
(
(
(
100 MHz, CDCl
CH), 128.45 (CH), 127.85 (CH), 127.80 (CH), 127.42 (CH), 126.99
CH), 125.51 (CH), 119.26 (C), 25.65 (3 × CH ), 25.55 (CH ), 23.30
); IR: 3013, 2963, 2932,
246 cm ; HRMS: Exact mass calc for C19 27N 269.2144, found:
69.2124.
3
): δ 132.08 (CH), 131.48 (CH), 128.74 (CH), 128.64
), 24.67 (CH
2
), 21.50 (CH
2
), 14.68 (CH
3
−1
3500–2500 (broad), 3012, 2963, 1729 cm ; HRMS: Exact mass calc
2
2
for C21
H
32
O 332.2351, found: 332.2369. The spectral data of 5 were in
3
CH
2
), 20.57 (CH
2
), 17.51 (CH
2
), 14.27 (CH
3
accord with those already published [29].
−
1
2
2
H
4.6. Biological evaluations
4.4. (4Z,7Z,10Z,13Z,16Z)-nonadeca-4,7,10,13,16-pentaen-1-ol (10)
4
.6.1. 3-oxa n-3 DPA conversion to monohydroxylated products
Nitrile 8 (0.28 g, 1.0 mmol) was dissolved in hexane (5 mL) and
3-oxa n-3 DPA (5, 0.3, 1, 10, 30 or 100 μM) was incubated with
cooled to −78 °C. DIBAL-H (1 M in hexane, 1.4 mL, 1.4 mmol) was
added dropwise. One hour later, more DIBAL-H (1 M in hexane, 0.7 mL,
soybean 15-LOX (Borate buffer, 4 °C, pH 9.2) for 17-OH-oxa n-3 DPA,
mouse recombinant 12-LOX (Tris-HCl 0.1 M, pH8, EDTA 5 mM) for 14-
OH-oxa n-3 DPA, human 5-LOX (Phosphate buffer 0.1 M, 0.08% Tween-
20, pH6.3) for 7-OH-oxa n-3 DPA or human recombinant COX-2 (Tris-
HCl 0.1 M, hematin porcine 20 μM, liquid phenol 0.67 mM, pH8) for
13-OH-oxa n-3 DPA. After 45 min the reaction was quenched using 2
volumes ice-cold methanol, products reduced using sodium borohy-
dride, and extracted using C18 SPE [30]. Products were isolated using
UV-RP-HPLC (Infinity 1260; Agilent Technologies). Online UV was
obtained for each. Here, an Agilent Poroshell 120 EC-C18 column
(100 mm × 4.6 mm × 2.7 μm) was kept at 50 °C and products isolated
with a mobile phase consisting of methanol-water-acetic acid of
50:50:0.01 (vol/vol/vol) that was ramped to 80:20:0.01 (vol/vol/vol)
from 2 min to 11 min, maintained till 14.5 min and then rapidly ramped
to 98:2:0.01 (vol/vol/vol) for the next 0.1 min. This was subsequently
maintained at 98:2:0.01 (vol/vol/vol) for 5.4 min, and the flow rate
was maintained at 0.5 mL/min.
0
.7 mmol) was added. After a total reaction time of three hours, the
reaction was quenched by addition of 1.4 M HCl to pH 2. The solution
was left stirring for another 30 min at −78 °C, then allowed to warm to
room temperature. Extraction with hexane (4 × 20 mL), drying
(
Na
2
4
SO ) and removal of solvents under reduced pressure, gave the
corresponding aldehyde of 9 (0.24 g; 83%) as a colorless oil. The al-
dehyde proved to be labile and was thus dissolved in MeOH (2.6 mL)
and cooled to 0 °C. Then NaBH
4
(0.085 g, 2.24 mmol) in MeOH
(
(
3.5 mL) was added. After 30 min, the reaction was quenched with HCl
1.4 M, 3.5 mL). The mixture was extracted with hexane: Et O (2:1) and
) and con-
2
the organic layer was washed with brine, dried (Na
SO
2 4
centrated under reduced pressure. The crude oil was purified by column
chromatography (silica gel; hexane:EtOAc 95:5) to afford alcohol 9
1
(
(
0.14 g, 0.51 mmol; 51% over two steps) as a clear oil. H NMR
400 MHz, CDCl
3
): δ 5.45–5.28 (m, 10H), 3.65 (t, J = 6.48 Hz, 2H),
2
.86–2.79 (m, 8H), 2.17 (q, J = 7.4 Hz, 2H), 2.07 (quint, J = 7.8 Hz,
6