Journal of Natural Products
Article
1
methanol to give 3 as a yellow solid (83 mg, 40% from Boc-Dap): H
washed with brine, dried (MgSO ), and concentrated to a crude solid,
4
NMR (CDCl , 400 MHz) δ 9.2 (1H, bs), 8.89 (1H, bs), 6.93 (1H, s),
0.105 g. Separation by flash silica gel chromatography (eluent:
3
.55 (1H, s), 6.54 (1H, s), 6.52 (1H, s), 5.73 (1H, bd, J = 9.8 Hz),
.97 (2H, m), 3.90 (3H, s), 3.87 (1H, m), 3.82 (3H, s), 3.79 (9H, s),
.73−3.59 (3H, m), 3.50 (1H, m), 3.42 (1H, m), 3.33 (1H, m), 3.21
CH Cl −CH OH, 9:1) gave 6 as a yellow foam solid (59 mg, 57%),
2
2
3
which was further purified on Sephadex LH-20 (eluent: CH OH):
3
1
TLC R 0.53 (CH Cl −CH OH, 9:1); H (CDCl , 400 MHz) δ 6.95
(1H, s), 6.54 (2H, m), 6.52 (1H, s), 5.81 (1H, dd, J = 12.2, 3.3 Hz),
f
2
2
3
3
5.29 (1H, m, NH), 3.92 (3H, s, OCH ), 3.87 (1H, m), 3.82 (3H, s,
3
OCH ), 3.81 (6H, s, 2 × OCH ), 3.47−3.36 (3H, m), 3.17−3.00
3
3
13
(5H, m), 2.89−2.82 (1H, m), 2.75−2.60 (4H, m), 2.56−2.46 (2H,
3
m), 2.24−2.06 (2H, m), 1.69−1.54 (1H, m), 1.45−1.26 (11H, m),
1.33 (s, 3 × CH ), 1.23−1.15 (2H, m), 1.14−1.02 (1H, m), 0.84 (3H,
3
1
3
t, J = 7.3 Hz); C (CDCl , 400 MHz) δ 170.7, 156.3, 148.0, 147.99,
3
147.8, 147.7, 130.5, 129.6, 125.9, 124.8, 111.7, 111.5, 110.2, 108.8,
79.4, 63.4, 61.4, 56.3, 56.2, 56.15, 56.12, 52.4, 49.2, 41.4, 39.6, 38.8,
37.9, 37.0, 36.8, 33.5, 29.0, 28.8, 28.6 (× 3C), 23.6, 11.1;
3
8
57
3
6
2
10c
+
(+)-HRESIMS m/z 652.3972 [M + H] (calcd for C H N O ,
3
7
54
3
7
0
.147 mmol) in anhydrous CH Cl (2 mL), under N at 0 °C, were
652.3962).
2
2
2
added TEA (85 μL, 0.063 g, 4.3 equiv) and DEPC (30 μL, 0.198
mmol, 1.37 equiv). The reaction mixture was stirred for 7 h with
warming to rt, then concentrated under reduced pressure to remove
the solvent and dried under a high vacuum overnight. Separation
using column chromatography and a gradient elution (EtOAc 100%
2′-N-(β-Alanine)-emetine TFA Salt (7). A solution of amide 6
(0.030 g, 0.046 mmol) in dry CH Cl (1 mL) was stirred and cooled
2
2
to 0 °C under N . TFA (0.20 mL) was added, and the reaction
2
mixture stirred for 2 h at rt; then the solvent was removed under
reduced pressure, and toluene was added as an azeotrope to further
concentrate and remove any residual TFA. The residue was purified
→
EtOAc−CH COCH (1:1) → ETOAc−MeOH (98:2 → 90:10)
3
3
followed by a CH Cl −MeOH (90:10) column wash gave the crude
product with TEA contamination. The crude product was dissolved in
CH Cl and extracted with water. The organic fraction was separated,
on Sephadex LH-20 (eluent: CH OH) to give 7 as a light yellow
3
2
2
1
colored solid (16 mg, 53% yield): H (CDCl
s), 6.57 (2H, s), 6.50 (1H, s), 5.68 (1H, d, J = 12 Hz), 4.21 (1H, m),
3.85, 3.83, 3.81 (3H each, OCH ), 3.64 (1H, s), 3.58 (1H, m), 3.45−
, 400 MHz) δ 6.83 (1H,
3
2
2
dried (MgSO ), and concentrated under reduced pressure to give the
3
4
product as an off-white/yellowish amorphous powder (108 mg; 71%
yield): TLC R 0.36 (CH Cl −MeOH, 91:9). This material was
3.37 (2H, m), 3.24−2.99 (6H, m), 2.97−2.82 (3H, m), 2.78 (1H, s),
2.76−2.68 (2H, m), 2.22 (1H, t J = 11.6 Hz), 1.75−1.53 (3H, m),
1.52−1.39 (2H, M).
f
2
2
further purified on a Sephadex LH-20 column (eluent: CH OH) to
3
give 5 as an amorphous yellow solid: TLC R 0.47 (CH Cl −MeOH,
2′-N-(Dov-Val-Dil-Dap-β-Ala)-Emetine (9). To a solution of
f
2
2
1
10a,b
8
%); mp 110−112 °C; H NMR (CDCl , 400 MHz) (conformational
isomers appeared to be present in solution as the signal assigned to H-
Boc-Dap,
(0.16 g, 0.54 mmol) in anhydrous DMF (4 mL) was
3
added TEA (76 μL, 0.06 g, 0.54 mmol), followed by HBTU (0.41 g,
1.08 mmol). The reaction mixture was stirred for 5 min before adding
a solution of 7 (0.3 g, 0.34 mmol) in DMF (1 mL). The solution was
1
′ on emetine appears as two signals in a 1:0.3 ratio) δ 7.09 (1H, s),
7
.00 (0.3H, m), 6.90 (1H, d, J = 9 Hz), 6.82−6.75 (0.6H, m), 6.63
(
0.3H, s), 6.61 (0.3H, d, J = 4.25), 4.78 (2H, m), 4.52 (0.3H, t, J = 8.4
stirred under N
2
for 16 h. Ethyl acetate (50 mL) was added, and the
organic layer was extracted with brine (2 × 50 mL), dried (MgSO ),
4
Hz), 4.21−4.11 (3H, m), 4.08 (3H, s, OCH ), 3.98 (2H, m), 3.88−
3
.81 (11H, m), 3.85, 3.84, 3.83 (3 × Ar-OCH ), 3.62 (1.6H, m),
and concentrated under reduced pressure to a crude product.
3
.51−3.35 (8H, m), 3.40, 3.32 (2 × OCH ), 2.99 (3H, s, NCH ),
Separation was carried out using flash column chromatography on
3
3
.93−2.82 (3H, m), 2.80 (1H, s), 2.78−2.69 (2H, m), 2.69−2.53
silica gel with a gradient elution system (CH
95:5) to give 2′-N-(boc-dap-β-ala)-emetine (8) as a yellow foam solid
(0.12 g, 48% yield): R 0.47 (CH Cl −MeOH, 94:6); (+)-HRESIMS
m/z 843.4894 [M + Na] (calcd for C46 Na, 843.4879).
Compound 8 was deprotected with TFA (0.2 mL, 2.53 mmol, 21
equiv) in CH Cl (2.5 mL) for 2 h at rt and concentrated with
toluene as an azeotrope under reduced pressure to give the TFA salt.
The salt was dissolved in CH Cl (5 mL), and the solution cooled to
2 2
Cl −MeOH, 97:3 →
13
3
f
2
2
+
400 MHz) δ 173.5, 173.2, 171.9, 169.6, 148.2, 147.9, 147.8, 131.5,
124.2, 111.5, 111.2, 110.3, 108.9, 86.8, 82.6, 77.4, 76.7, 62.0, 61.2,
60.6, 59.6, 59.2, 58.3, 56.9, 56.3, 56.1, 53.9, 53.6, 51.8, 48.5, 47.8,
43.1, 40.4, 39.7, 39.4, 38.7, 37.9, 35.9, 33.4, 32.1, 31.2, 31.1, 29.9,
29.5, 27.9, 25.9, 25.2, 23.7, 23.5, 20.3, 19.8, 18.3, 18.0, 16.4, 16.0,
1
5.7, 11.0; (+)-HRESIMS m/z 1061.7300 [M + H]+ (calcd for
H N O
68 4 9
2
2
2
2
10c
0 °C (ice bath). Dov-Val-Dil-TFA salt was then added followed by
DEPC (22 μL, 0.144 mmol, 1.2 equiv) and TEA (0.42 g, 58 mL, 3.5
equiv). The reaction mixture was stirred at 0 °C for 6 h and
monitored by TLC (CH Cl −MeOH, 90:10), then concentrated
C H N O , 1061.7261).
60
97
6
10
2
′- (N-Carboxy-β-alanyl)-tert-butyl Emetine Ester (6). N-Boc-
β-alanine. A solution of β-alanine (0.2 g, 22.4 mmol) in 1 M NaOH−
THF (1:2) (30 mL) was cooled to 0 °C. Di-tert-butyldicarbonate (4.9
g, 22.4 mmol) was added, and the reaction mixture stirred with
warming to rt for 16 h. The solvent was removed under reduced
pressure, and the aqueous layer acidified to pH 2.0 with 4 M HCl and
extracted with diethyl ether (5 × 10 mL). The combined organic
layers were washed with brine, dried (Na SO ), filtered, and
2
2
under reduced pressure and dried further overnight under a high
vacuum. The crude product was separated by column chromatog-
raphy using gradient elution (CH Cl 100% → CH Cl −MeOH,
2
2
2
2
90:10) to give the product still contaminated with TEA. The product
mixture was then dissolved in CH Cl , extracted with water, and dried
2
2
(MgSO ) under a high vacuum, to give the product 9 as an off-white
2
4
4
concentrated under reduced pressure, to give N-Boc-β-alanine as a
amorphous solid (27 mg, 20% yield): R 0.14 (CH Cl −MeOH,
f
2
2
1
white crystalline solid, which was collected using hexane and dried
93:7); H (CDCl , 400 MHz) (conformers observed) δ 6.94 (1H, s),
3
3
1
(
2.0 g, 51% yield): mp 70 °C [lit. 71−72 °C]; H NMR (CDCl , 400
6.92 (1H, m, NH), 6.73 (1H, t, J = 5.9 Hz, NH)), 6.57 (1H, s), 6.55
(1H, s), 6.53 (1H, s), 5.77 (1H, dd, J = 11.8, 3.4 Hz), 4.75 (1H, dd, J
3
MHz) δ 11.19 (1H, bs, OH), 6.26, 5.13 (1H, bs, NH), 3.40 (2H, m),
.58 (2H, m), 1.44 (9H, s); C NMR (CDCl , 400 MHz) δ 177.6,
55.9, 79.8, 35.9, 34.5, 28.4. N-Boc-β-alanine (0.03 g, 0.158 mmol)
1
3
2
1
= 9.6, 6.6 Hz), 4.10 (2H, m), 3.94 (3H, OCH ), 3.93 (1H, m), 3.86
3
3
(1H, m), 3.84 (3H, s, OCH ), 3.82 (6H, s, OCH ), 3.77−3.64 (2H,
3
3
was dissolved in anhydrous DMF (1 mL) at rt under N . HBTU (0.12
m), 3.49−3.35 (4H, m), 3.33 (3H, s, OCH ), 3.29 (3H, s, OCH ),
2
3
3
g, 0.316 mmol, 2 equiv) followed by DIEA (0.027 mL, 0.020 mg,
3.30 (1H, m), 3.01 (3H, s, NCH ), 2.95−2.81 (2H, m), 2.79−2.50
3
0
.158 mmol) were added with stirring for 10 min. Emetine
(7H, m), 2.45 (2H, m), 2.38 (1H, m), 2.36−2.20 (8H, m), 2.25 (s,
dihydrochloride (1) (0.09 g, 0.158 mmol) was then added to the
reaction mixture. Stirring was continued for 20 h at rt, and the
reaction was monitored by TLC (CH Cl −CH OH, 9:1). Ethyl
NCH × 2), 2.12−1.89 (4H, m), 1.76 (1H, m), 1.60 (2H, m), 1.40−
3
1
3
0.77 (CH, CH , CH overlapping peaks); C NMR (CDCl , 400
2
3
3
MHz) (conformers observed) δ 174.4, 173.9, 173.6, 171.8, 170.8,
170.6, 170.4, 170.1, 148.13, 148.0, 147.99, 147.9, 147.8, 130.4, 130.2,
2
2
3
acetate (10 mL) was added, and the organic layer separated and
D
J. Nat. Prod. XXXX, XXX, XXX−XXX