Antimicrobial Activities of Naphthazarins
J ournal of Natural Products, 2002, Vol. 65, No. 12 1861
Extr a ction a n d Isola tion . The whole air-dried plant (10
kg) was extracted with 95% ethanol (50 L) three times at 60
°C for 24 h. The ethanolic extracts were combined and
concentrated in vacuo to 2 L. The concentrated extract was
suspended in H2O (10 L) and then extracted with EtOAc (3 ×
10 L). After evaporation of EtOAc, the concentrated mixture
was mixed with 150 g of silica gel (230-400 mesh). The air-
dried mixture was subjected to a chromatographic column (4
× 100 cm) and then eluted with hexane and 20%, 40%, 60%,
80%, and 100% CHCl3/hexane, followed by 5% MeOH/CHCl3
(4 L each). Fractions (500 mL each) were collected, and similar
fractions were combined. The combined fractions 15-19, 22-
27, and 31-37 showed antimicrobial activity. Fractions 15-
19 were rechromatographed by PTLC using 50% CHCl3/hexane
as the developing solvent to yield isobutyryl alkannin (5)22 (19
mg) and a mixture of 6, 7, and 8 (55 mg). The mixture 6-8
was further chromatographed on a silica gel column (1 × 120
cm) with hexane and 3%, 5%, and 8% EtOAc/hexane (600 mL
each) as the eluent to yield â,â-dimethylacryloyl alkannin (6)23
(32 mg), isovaleryl alkannin (7)24 (11 mg), and (S)-R-methyl-
butyryl alkannin (8) (4 mg). Fractions 22-27 were purified
on a silica gel column (1 × 120 cm) with 10% and 20% CHCl3/
hexane (600 mL each) as the eluent to yield a mixture of acetyl
alkannin (3)22 and acetyl shikonin (4)22 (33 mg). The mixture
of 3 and 4 was further separated by HPLC with a chiral phase
column using 2.5% 2-propanol/hexane (v/v) as mobile phase
to obtain two peaks with retention time of 15.58 and 14.26
min, respectively. Fractions 31-37 were rechromatographed
on a silica gel column (1 × 120 cm). Elution with 20%, 25%,
30%, 35%, and 40% CHCl3/hexane (600 mL each) gave hope-
none-I (10)27 (10 mg), ergosta-4,6,8(14),22-tetraen-3-one (11)28
(18 mg), stigmast-4-ene-3,6-dione (12)29 (3 mg), tetracosyl
ferulate (13)30 (4 mg), hexacosyl ferulate (14)31 (7 mg), octacosyl
ferulate (15)32 (5 mg), and a mixture of alkannin (1)22,25 and
shikonin (2)22,25 (65 mg). The mixture of 1 and 2 was further
separated by a chiral HPLC eluted with 10% 2-propanol/
hexane (v/v) to result in two peaks with retention times of
19.92 and 14.80 min, respectively.
Hop en on e-I (10): colorless solids; mp 195-197 °C (lit.27
197-199 °C); 1H NMR (CDCl3, 500 MHz) δ 0.83 (3H, s, H-28),
0.90 (3H, d, J ) 7.0 Hz, H-29), 0.91 (3H, s, H-25), 0.96 (3H, s,
H-26), 0.97 (3H, d, J ) 7.0 Hz, H-30), 1.01 (3H, s, H-24), 1.03
(3H, s, H-27), 1.07 (3H, s, H-23), 1.28 (1H, m, H-19â), 1.65
(1H, dd, J ) 7.0, 12.0 Hz, H-19R), 1.91 (1H, m, H-16ax), 1.93
(1H, m, H-1eq), 2.10 (1H, dd, J ) 9.5, 15.5 Hz, H-20â), 2.18
(1H, m, H-20R), 2.26 (1H, dt, J ) 14.0, 3.5 Hz, H-16eq), 2.41
(1H, ddd, J ) 5.0, 8.0, 16.0 Hz, H-2eq), 2.48 (1H, ddd, J ) 8.0,
10.0, 16.0 Hz, H-2ax), 2.62 (1H, sept, J ) 7.0 Hz, H-22); 13C
NMR (CDCl3, 125 MHz) δ 218.6 (s, C-3), 140.0 (s, C-17), 136.5
(s, C-21), 55.1 (d, C-5), 50.4 (d, C-9), 50.0 (s, C-18), 49.7 (d,
C-13), 47.6 (s, C-4), 42.3 (s, C-14), 42.0 (t, C-19), 41.9 (s, C-8),
40.0 (t, C-1), 37.1 (s, C-10), 34.4 (t, C-2), 33.0 (t, C-7), 32.1 (t,
C-15), 27.7 (t, C-20), 26.9 (q, C-23), 26.6 (d, C-22), 24.3 (t, C-12),
22.2 (t, C-11), 22.1 (q, C-29), 21.5 (q, C-30), 21.3 (q, C-24), 20.0
(t, C-6, C-16), 19.3 (q, C-28), 16.4 (q, C-26), 16.3 (q, C-25), 15.1
(q, C-27); EIMS m/z 424 [M]+ (17), 409 (14), 381 (100), 202
(9), 188 (10), 175 (11), 161 (31), 148 (12), 133 (23), 119 (16).
Stigm a st-4-en e-3,6-d ion e (12): colorless solids; mp 57-
59 °C (lit.29 60-64 °C); 1H NMR (CDCl3, 500 MHz) δ 0.71 (3H,
s, H-18), 0.81 (3H, d, J ) 6.5 Hz, H-26), 0.83 (3H, d, J ) 6.5
Hz, H-27), 0.85 (3H, t, J ) 7.0 Hz, H-29), 0.92 (3H, d, J ) 6.5
Hz, H-21), 1.15 (3H, s, H-19), 1.50 (1H, dt, J ) 4.0, 13.0 Hz,
H-11ax), 1.58 (1H, m, H-15), 1.64 (1H, m, H-11eq), 1.67 (1H,
m, H-25), 1.90 (3H, m, H-1ax, H-8, H-16â), 2.02 (1H, dd, J )
12.5, 16.0 Hz, H-7â), 2.10 (1H, m, H-12eq), 1.91 (1H, m,
H-16ax), 1.93 (1H, m, H-1eq), 2.44 (1H, m, H-2ax), 2.52 (1H,
dt, J ) 5.0, 16.0 Hz, H-2eq), 2.67 (1H, dd, J ) 4.0, 16.0 Hz,
H-7R); 13C NMR (CDCl3, 125 MHz) δ 202.3 (s, C-6), 199.5 (s,
C-3), 161.1 (s, C-5), 125.4 (d, C-4), 56.5 (d, C-14), 55.9 (d, C-17),
51.0 (d, C-9), 46.8 (t, C-7), 45.8 (d, C-24), 42.5 (s, C-13), 39.8
(s, C-10), 39.1 (t, C-12), 36.0 (d, C-20), 35.5 (t, C-1), 34.2 (d,
C-8), 34.0 (t, C-2), 33.8 (t, C-22), 29.1 (d, C-25), 28.0 (t, C-16),
26.0 (t, C-23), 24.0 (t, C-15), 23.1 (t, C-28), 20.9 (t, C-11), 19.8
(q, C-27), 19.0 (q, C-26), 18.7 (q, C-21), 17.5 (q, C-19), 12.0 (q,
C-29), 11.9 (q, C-18).
Cin n a m oyl a lk a n n in (16): red solid; UV (CH3OH) λmax (log
ꢀ) 215 (4.56), 278 (4.35), 487 (3.67), 518 (3.70), 558 (3.46) nm;
IR (film) νmax 2917, 1713, 1632, 1608, 1571, 1492, 1445, 1240,
P r ep a r a tion of (R)-(-)-2-Meth ylbu tyr ic Acid . DL-2-
Methylbutyryl chloride was esterified with (S)-2-hydroxy-3-
phenylpropionic acid in the presence of triethylamine and
4-(dimethylamino)pyridine (DMAP). The resulting (S)-2-[2-
methylbutyryloxy]-3-phenylpropionic acid was treated with
oxalyl chloride in 1% DMF/CH2Cl2 and converted to (S)-N-
methyl-2-[2-methylbutyryloxy]-3-phenylpropionamide with me-
thylamine in THF. The amide diasteroisomers were separated
by silica gel column chromatography to give (S)-N-methyl-2-
[(R)-2-methylbutyryloxy]-3-phenylpropionamide, which was
subsequently hydrolyzed to yield optically pure (R)-(-)-2-
methylbutyric acid.42
1
1203, 1161, 1035 cm-1; H and 13C NMR, see Tables 1 and 2;
EIMS m/z 462 [M]+ (1), 249 (3), 270 (98), 255 (100), 237 (12),
227 (17), 191 (23), 175 (66), 145 (42), 117 (19), 89 (19).
3,4-(Meth ylen edioxy)cin n am oyl alkan n in (17): red solid;
UV (CH3OH) λmax (log ꢀ) 214 (4.68), 286 (4.18), 330 (4.24), 487
(3.80), 518 (3.83), 560 (3.61) nm; IR (film) νmax 2917, 1715,
1632, 1609, 1575, 1453, 1333, 1263, 1231, 1200, 1157, 768 cm-
1
1; H and 13C NMR, see Tables 1 and 2; EIMS m/z 418 [M]+
(2), 253 (5), 270 (98), 255 (100), 237 (17), 229 (23), 199 (20),
171 (13), 147 (56), 131 (21), 103 (18), 87 (24), 77 (8).
Gen er a l Syn th eses of Alk a n n in Ester s. To a solution of
alkannin (20 mg) in anhydrous CH2Cl2 (10 mL) were added
dicyclohexylcarbodiimide (30 mg), DMAP (4 mg), and the
corresponding carboxylic acid (0.07 mmol) under nitrogen
atmosphere in an ice bath. The reaction mixture was stirred
in the ice bath for 3 h, and the stirring mixture was kept at
room temperature overnight. The resulting mixture was
diluted with hexane (20 mL) and filtered. The filtrate was
concentrated in vacuo and purified by PTLC. The pure
alkannin esters were obtained in 8-15% yield.
Ba cter ia . The methicillin-resistant Staphylococcus aureus
(MRSA 8-8S), vancomycin-resistant Enterococcus faecium
(VRE F935), and vancomycin-resistant Enterococcus faecalis
(VRE CKU-17) were isolated from sputum, urine, and pus of
patients, respectively. MRSA 8-8S and VRE F935 were col-
lected from the clinical microbiology laboratory of the Tri-
Service General Hospital, Taipei, while VRE CKU-17 was
collected from National Cheng-Kung University Medical Col-
lege, Taiwan.
An tiba cter ia l Activity. Antibacterial activity of crude
extracts or fractions against microorganisms were screened
using the paper disk method on Luria-Bertani (LB) agar plates
according to the National Committee for Clinical Laboratory
Standards (NCCLS).43 The MICs for the active components
were then determined by the broth microdilution method.44
Briefly, the test compounds were dissolved in DMSO, which
was less than 5% for all the final tests. Subcultured microor-
ganisms were diluted as recommended44 and added to wells
containing 0.1 mL of Mueller-Hinton (MH) (Staphylococcus)
or LB (all other bacteria) broth with 2-fold serial dilutions of
the test compounds, to yield the final inoculum of approxi-
mately 5 × 105 CFU/mL. The actual inoculum was quantita-
tively measured on an LB agar plate and served as MBC
control. The MICs were determined after 24 h of incubation
at 35 °C with ambient air. The MIC was defined as the lowest
(S)-r-Meth ylbu tyr yl a lk a n n in (8): red solid; UV (CH3-
CN) λmax (log ꢀ) 192 (4.93), 216 (4.51), 277 (3.97), 488 (3.39),
520 (3.47), 559 (3.20) nm; IR (film) νmax 2959, 2922, 1727,
1
1461, 1272, 1122, 1071 cm-1; H and 13C NMR, see Tables 1
and 2; EIMS m/z 372 [M]+ (2), 288 (5), 270 (100), 255 (83),
237 (10), 227 (8), 220 (18); HREIMS m/z 372.1550 (calcd for
C
21H24O6, 372.1567); CD (c 0.144, CH3OH) θ (nm) -680 (230),
-3625 (250), 0 (288), +2334 (307), 0 (328), -3640 (356), -715
(398).
(R)-r-Meth ylbu tyr yl a lk a n n in (9): red solid; 2.1 mg; UV
(CH3OH) λmax (log ꢀ) 214 (4.45), 275 (3.76), 487 (3.65), 518
(3.68), 559 (3.45) nm; IR (film) νmax 2960, 2923, 1727, 1460,
1
1271, 1122, 1071 cm-1; H and 13C NMR, see Tables 1 and 2;
CD (c 0.130, CH3OH) θ (nm) -1243 (234), -3503 (250), 0 (288),
+2564 (307), 0 (329), -3687 (356), -402 (398).