Antineoplastic agents. 527. Synthesis of 7-deoxynarcistatin, 7-deoxy-trans-dihydronarcistatin, and trans-dihydronarcistatin 1

Article abstract of DOI:10.1021/np0304518

The synthesis of sodium narcistatin (8) was improved (88% overall yield) and the modified reaction sequence was utilized to synthesize three new 3,4-cyclic phosphate prodrugs, sodium 7-deoxynarcistatin (5), sodium 7-deoxy-trans-dihydronarcistatin (6), and

Full text of DOI:10.1021/np0304518

J. Nat. Prod. 2005, 68, 207-211
207
Antineoplastic Agents. 527. Synthesis of 7-Deoxynarcistatin,
7-Deoxy-trans-dihydronarcistatin, and trans-Dihydronarcistatin 11
George R. Pettit* and Noeleen Melody
Cancer Research Institute and Department of Chemistry and Biochemistry, Arizona State University,
Tempe, Arizona 85287-2404
Received October 16, 2003
The synthesis of sodium narcistatin (8) was improved (88% overall yield) and the modified reaction
sequence was utilized to synthesize three new 3,4-cyclic phosphate prodrugs, sodium 7-deoxynarcistatin
(5), sodium 7-deoxy-trans-dihydronarcistatin (6), and sodium trans-dihydronarcistatin (7). The human
cancer cell line inhibitory isocarbostyril precursors were isolated from the bulbs of Hymenocallis littoralis
obtained by horticultural production or reduction of narciclasine (1a f 4) from the same source.
Advances in the chemistry of isocarbostyrils were already
being achieved in the 1909-1920 period by the well-known
organic chemists Dieckmann,2a Robinson,2b Perkin,2b and
Chichibabin.2c However, interest in the potential of natu-
rally occurring isocarbostyrils as anticancer agents only
began with the isolation of narciclasine (1a)3-8 in 1967 and
our cancer bioassay-directed discovery9 of pancratistatin
(2) as a constituent of Pancratium littorale (now Hymeno-
callis littoralis, Amaryllidaceae) accompanied by narci-
clasine (1a) and 7-deoxynarciclasine (1b, lycoricidine,
margetine). Later, we isolated another new anticancer
constituent from H. littoralis identified as 7-deoxy-trans-
dihydronarciclasine (3a).10a These discoveries and the need
for scale-up quantities of this series of important isocar-
bostyrils for preclinical development led us to use a
biotechnological approach involving an initial tissue culture
cloning of H. littoralis bulbs.10,11a,b Large numbers of H.
littoralis bulbs10 were then grown in Arizona specifically
for production of (+)-pancratistatin (2) and narciclasine
(1a), along with 7-deoxynarciclasine (1b) and 7-deoxy-
trans-dihydronarciclasine (3a). The pancratistatin (2) and
narciclasine (1a) constituents were used for subsequent
structural modifications,12 conversion to phosphate pro-
drugs,13 and anticancer evaluations.10-14 A 7-deoxynarci-
clasine (1b)/7-deoxy-trans-dihydronarciclasine (3a) fraction
provided the starting material for analogous objectives that
included the present study.
1b and 3a in 72% yields. The 3,4-cyclic phosphates 5, 6,
and 74,9c were synthesized employing an improvement in
the procedure we developed for synthesis of sodium nar-
cistatin (8)13a using tetrabutylammonium dihydrogen phos-
phate and an excess of dicyclocarbodiimide in dry pyridine
under argon at 80 °C for 48 h.
1
In each case, H NMR of the crude product showed the
reaction to be only 50% complete following a 24 h period.
Additional reagents were added at this stage, and the
reaction was allowed to proceed to completion (a further
24 h). Increasing the amount of tetrabutylammonium
dihydrogen phosphate from 0.65 equiv to 1 equiv in the
first 24 h did not increase the reaction rate. Water was
added to the reaction mixture to precipitate the dicyclo-
hexylurea (DCU), and the pyridine/water filtrate was
concentrated to remove the pyridine. An aqueous extract
of the residue was passed through a Dowex 50WX8-400
ion-exchange column (sodium form). The UV-responsive
fractions were combined and lyophilized to afford the new
3,4-cyclic phosphates designated sodium 7-deoxynarcistatin
(5, 88% yield), sodium 7-deoxy-trans-dihydronarcistatin (6,
65% yield), and sodium trans-dihydronarcistatin (7, 94%
yield). More recently,17 we found that deletion of the
p-toluenesulfonic acid component increased the yield of
phosphate 8 to 88% versus the original13a 50%. That was
one of the major improvements that allowed new phos-
phates 5, 6, and 7 to be obtained in very good yields.
Because isocarbostyrils 1b, 3a, and 44,9c also exhibited
strong cancer cell growth inhibition against a minipanel
of human cancer cell lines (Table 1), they were next selected
for conversion to 3,4-cyclic phosphate prodrugs13a for the
purpose of improving aqueous solubility and transport to
tumors for cancer antiangiogenesis/vascular targeting.15,16
Detailed analyses of the 1H, 13C, COSY, HMQC, HMBC,
and D2O exchange spectra were conducted in an effort to
assign the carbon and proton spectra of each of the cyclic
phosphates and confirm the structure assignments. The 31
P
NMR (DMSO-d6 with neat H3PO4 as external standard)
spectra corresponding to phosphates 5, 6, and 7 showed
downfield signals for the phosphorus atom at δ 15.23,
13.45, and 13.36, respectively, which are consistent with
the data previously observed for cyclic phosphate 8.13a In
addition, sodium narcistatin (8), prepared by the procedure
described herein, was found to be identical (mp, 1H NMR,
31P NMR) with an authentic sample.13a
Downfield shifts for the H-3 and H-4 resonances dis-
played by 3,4-cyclic phosphates 5-8 were observed in all
cases when compared with the spectra of each of the
starting isocarbostyrils. The 1H NMR spectrum of 7-deoxy-
narciclasine (1b) revealed the H-3 and H-4 resonances at
δ 3.69 and 3.76, respectively, whereas the derived phos-
phate (5) showed H-3 and H-4 downfield as a multiplet at
δ 3.93-3.79. In trans-dihydro derivative 3a, the multiplet
Results and Discussion
The 7-deoxynarciclasine (1b) and 7-deoxy-trans-dihydr-
onarciclasine (3a) mixture separated from H. littoralis was
acetylated with acetic anhydride in pyridine and the
peracetate mixture separated by silica gel column chro-
matography to afford isocarbostyrils 1c (60% recovery) and
3b (19% recovery). Both 2,3,4-triacetoxy-7-deoxynarci-
clasine (1c) and 2,3,4-triacetoxy-7-deoxy-trans-dihydronar-
ciclasine (3b) were then deprotected with potassium car-
bonate in aqueous MeOH to afford the corresponding triols
* To whom correspondence should be addressed. Tel: (480) 965-3351.
Fax (480) 965-8558.
10.1021/np0304518 CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/28/2005

208 Journal of Natural Products, 2005, Vol. 68, No. 2
Pettit and Melody
Table 1. Aqueous Solubility, Human Cancer Cell Line, and Murine P-388 Lymphocytic Inhibitory Activities
ED50 (µg/mL)
GI50 (µg/mL)
aqueous solubility
25 °C (mg/mL)
leukemia
P388
pancreas-a
BXPC-3
breast
MCF-7
CNS
SF268
lung-NSC
NCI-H460
colon
KM20L2
prostate
DU-145
isocarbostyril
1b
3a
4
<1
<1
0.019
0.029
0.0024
1.7
0.42
1.4
1.6
0.39
1.7
0.88
0.26
0.35
0.070
0.046
0.012
5.3
0.046
0.034
0.0053
4.0
0.120
0.059
0.020
6.3
0.053
0.043
0.0092
4.7
0.084
0.051
0.015
5.6
0.051
0.040
0.0066
3.9
<1
5
>190
>10
>10
>10
>5
5a
5b
6
6a
6b
7
>1
>1
>1
>1
>1
>1
>1
>10
>1
>1
5.6
>1
>1
>1
7.2
>1
>10
>1
>1
8.8
>1
>1
>1
>10
>1
>1
7.8
>1
>1
>1
>10
>1
>1
9.6
>1
>1
>1
>10
>1
>1
>1
>5
>1
>10
>1
4.6
5.2
7a
7b
>1
>1
>1
0.64
0.54
1
absent in the spectrum of each when a D2O exchange H
NMR experiment was performed. The D2O exchange
experiment also allowed assignment of the N-H reso-
nances for 3,4-cyclic phosphate 5 at δ 7.65, 6 at δ 7.53, and
7 at δ 8.40.
The 13C spectra were examined using HMQC and
HMBC. With 7-deoxynarciclasine (1b) f 3,4-cyclic phos-
phate 5, there was a downfield shift of the C-3 and C-4
signals from δ 69.9 to 75.6 and 74.8 (C-3, C-4). The
corresponding downfield shifts for 3a f 6 were δ 70.4 and
72.3 for C-3 and C-4, respectively, to δ 75.7 (C-3) and 75.9
(C-4), and for phosphate 7 C-3 and C-4 appeared at δ 74.9.
Analysis of the COSY spectrum for phosphate 5 indicated
the H-1 signal was downfield at δ 6.35 and showed strong
correlation peaks with the OH signal at δ 5.71 and the H-2
signal at δ 4.24. The H-4a signal was assigned to the
multiplet at δ 4.24. The COSY spectrum for phosphate 6
showed H-1ax at δ 1.47 ppm and H-1eq at δ 2.27 exhibiting
strong correlation peaks with the multiplet at δ 2.84 (H-
10b) and the H-2 resonance at δ 4.03. The H-10b proton
showed cross-peaks with the proton signal at δ 3.54, which
was assigned to H-4a. Cross-peaks were also observed
between H-4a and the H-4 signal in the multiplet at δ 4.03.
The H-3 resonance was therefore assigned to the narrow
multiplet at δ 4.11. An HMBC spectrum of phosphate 6
was used to assign the protons H-7 and H-10. The H-7
resonance (δ 7.27) showed cross-peaks with the C-6 car-
bonyl signal at δ 164.7, whereas the H-10 (δ 6.93) showed
cross-peaks with the C-10a signal at δ 124.2 and the C-10b
at δ 32.7. The proton spectrum from phosphate 7 was
analyzed using COSY in a similar manner. The resonances
for H-1ax and H-1eq were assigned at δ 1.50 and 2.26,
respectively, as expected. These protons showed strong
correlation peaks with the H-2 resonance in the multiplet
between δ 4.07 and 4.03 and the H-10b signal at δ 2.84.
Consequently, the H-4a signal was easily identified as a
doublet of doublets at δ 3.59. The narrow multiplet at δ
4.14 was assigned to H-3, as a strong correlation peak was
observed between the H-4a signal and the H-4 signal in
the multiplet at δ 4.07-4.03. These 2D NMR spectral
assignments for 3,4-cyclic phosphates 5, 6, and 7 were
consistent with those already reported for sodium narci-
statin, where the structure was unequivocally established
by X-ray crystallography.13a
at δ 3.69 was assigned to H-3 and H-4 and in cyclic
phosphate 6 they were at δ 4.11 (H-3) and 4.03 (H-4).
Similarly, the downfield signals for protons H-3 and H-4
appeared as part of a multiplet at δ 4.14-4.03 for phos-
phate 7. Furthermore, the usual signals for OH-3 and OH-4
The cyclic phosphate prodrugs 5, 6, and 7 along with the
parent compounds 1b, 3a, and 4 were evaluated against a
minipanel of human cancer cell lines and murine P388
lymphocytic leukemia (Table 1). Results of the cancer cell
line evaluations reconfirmed the strong cancer cell growth
inhibitory activity of 7-deoxy-trans-dihydronarciclasine (3a)
and trans-dihydronarciclasine (4). As expected, the corre-
1
were absent from the H NMR spectra of each of the 3,4-
cyclic phosphates. The OH-2 signals for 3,4-cyclic phos-
phates 5-7 were found downfield at δ 5.71, 5.22, and 5.26,
respectively. By way of verification, these signals were

Antineoplastic Agents
Journal of Natural Products, 2005, Vol. 68, No. 2 209
H-10b), 2.5 (1H, m, H-1eq), 2.07 (3H, s, -OCOCH3), 2.09 (3H,
s, -OCOCH3), 2.14 (3H, s, -OCOCH3), 1.94 (m, 1H, H-1ax);
13C NMR (DMSO-d6, 75 MHz) δ 169.8 (C, OCOCH3) 168.7 (C,
OCOCH3), 168.6 (C, OCOCH3), 165.3 (C, C-6), 150.9 (C), 146.5
(C), 135.2 (C), 122.6 (C), 107.7 (CH, C-7), 103.4 (CH, C-10),
101.3 (CH2, -OCH2O-), 70.9 (CH, C-4), 68.1 (CH, C-2), 67.0
(CH, C-3), 52.3 (CH, C-4a), 34.2 (CH, C-10b), 26.2 (CH, C-1),
20.5 (CH3, -OCOCH3), 20.4 (CH3, -OCOCH3), 20.2 (CH3,
-OCOCH3); HRAPCI+ m/z 420.1282 [M + H]+ (calcd for
C20H21NO9 420.1294 [M + H]+).
sponding 3,5-cyclic phosphates owing to comparatively slow
release of the parent active compound were less inhibitory
under the experimental conditions employed.13a However,
cleavage of the phosphate groups is expected to be very
effective in vivo,16 and such anticancer evaluations are now
underway as part of the further preclinical development
of these new anticancer drug candidates.
Experimental Section
General Experimental Procedures. Reagents were pur-
chased from Acros Chemical Company unless otherwise noted
and used as received. Solvents were distilled prior to use, and
pyridine was dried over KOH pellets and distilled. The
7-deoxynarciclasine, 7-deoxy-trans-dihydronarciclasine, and
trans-dihydronarciclasine were isolated10 from Hymenocallis
littoralis grown by the ASU-CRI research group in Tempe, AZ.
These isocarbostyrils were visible as blue fluorescent spots on
TLC plates under long-wave ultraviolet light and developed
with I2 vapor (see also ref 13a). Dowex 50WX8-400 cation-
exchange resin (H+ form) was first eluted with MeOH, 1 N
HCl, and deionized H2O. The cation forms of the resin were
prepared by eluting with a 1 N solution of the appropriate base
followed by deionized H2O.
7-Deoxynarciclasine (1b). To a solution of 2,3,4-triac-
etoxy-7-deoxynarciclasine (1c, 4.36 g) in CH3OH-(99 mL)
H2O-(1 mL) CH2Cl2 (30 mL) was added K2CO3 (0.124 g) and
stirring continued for 16 h at rt while a white precipitate
separated. The mixture was neutralized with acetic acid (2
mL), stirred for 15 min, and concentrated to minimum volume.
The colorless product was collected (2.18 g, 72%), and recrys-
tallization from acetic acid-MeOH afforded fine needles: mp
205-210 °C (dec), 230 °C melts; [R]23 +147° (c 0.52, DMSO)
D
[lit.9c mp 251-252°C, [R]33D +157.3° (c 0.96, DMSO)]; 1H NMR
(DMSO-d6, 300 MHz) δ 7.29 (1H, s, H-7), 7.24 (1H, s, H-10),
7.17 (1H, s, NH), 6.10-6.08 (3H, m, H-1, -OCH3O-), 5.15 (2H,
m, OH), 4.96 (1H, m, OH), 4.16 (1H, d, J ) 8.1 Hz, H-4a), 4.01
(1H, m, H-2), 3.76 (1H, m, H-4), 3.69 (1H, m, H-3); 13C NMR
(DMSO-d6, 75 MHz) 163.8 (C, C-6), 151.7 (C), 148.5 (C), 132.4
(C), 130.7 (C), 124.3 (C), 122.6 (CH, C-1), 106.9 (CH, C-7), 103.9
(CH, C-10), 102.5 (CH2, -OCH2O-), 73.2 (CH, C-1), 69.9 (2CH,
C-4, C-3), 53.4 (CH, C-4a); HRAPCI+ m/z 292.0770 (M + H)+
(calcd for C14H14NO6, 292.0891 [M + H]+).
Melting points were obtained using a Fisher-Johns melting
point apparatus and are uncorrected. Optical rotations were
measured using a Perkin-Elmer 241 polarimeter. Thin-layer
chromatography was conducted using Analtech silica gel
GHLF plates. High-resolution mass spectra were obtained by
the Washington University Mass Spectrometry Laboratory (St.
Louis, MO) and by a JEOL LCMate magnetic sector instru-
ment either in the FAB mode, with a glycerol matrix, or by
APCI with a poly(ethylene glycol) reference. All 1H NMR
spectra were initially obtained using a Varian Gemini 300
MHz instrument unless otherwise noted. The 13C, 1H-1H
Sodium 7-Deoxynarcistatin (5). A solution of 7-deoxy-
narciclasine (1b, 0.2 g, 0.69 mmol) in pyridine (8 mL) was
heated to 80 °C, and tetrabutylammonium dihydrogen phos-
phate (0.15 g, 0.45 mmol, 0.65 equiv) followed by dicyclohexy-
lcarbodiimide (0.8 g, 5.6 equiv) were added. The reaction was
1
allowed to proceed at 80 °C for 24 h. An H NMR analysis of
the reaction mixture composition indicated a 50:50 mixture
of starting material to product. Tetrabutylammonium dihy-
drogen phosphate (0.15 g) was added followed by DCCI (0.8
g), and the reaction continued for a further 24 h. At this point,
1H NMR analysis of a sample from the reaction mixture
showed the reaction was complete. The reaction mixture was
cooled, and H2O (100 mL) was added. The precipitated
dicyclohexylurea (DCU) was collected, and the pyridine-H2O
mother liquor was concentrated to minimum volume. The
aqueous fraction was then passed through an ion-exchange
column (DOWEX 50W8-400) in the sodium form. The UV-
responsive fractions were combined and lypholized to yield
1
1
COSY, H-13C HMBC, H-13C HMQC, and 31P NMR experi-
ments were conducted employing a Varian Inova 400 MHz
instrument.
Separation of 7-Deoxynarciclasine (1b) and 7-Deoxy-
trans-dihydronarciclasines (3a). The H. littoralis frac-
tions10 (8.6 g) containing isocarbostyrils 1b and 3a were
acetylated by dissolving in pyridine (20 mL) and adding acetic
anhydride (20 mL, 2.4 equiv). The mixture slowly became a
solution with stirring overnight at rt, and TLC (CH2Cl2-CH3-
OH, 2%) showed no starting material. Ice water (200 mL) was
added to the reaction mixture with vigorous stirring. A cream-
colored precipitate developed and was collected after stirring
for 2 h to provide 13.7 g. The acetylation product (9 g) was
separated on a silica gel column (37 cm × 7 cm) by elution
using 7:3 toluene-ethyl acetate to yield 2,3,4-triacetoxy-7-
deoxynarciclasine (1c) (5.45 g, 60.5%) followed by a mixture
of both 1c and 3b acetates (1.08 g) and finally 2,3,4-triacetoxy-
7-deoxy-trans-dihydronarciclasine (3b, 1.7 g, 19%). Recrystal-
lization of peracetate 1c from CH2Cl2-CH3OH gave colorless
phosphate 5 as a colorless solid: 227 mg (88%); mp 255°C (dec);
1
[R]24 -7.2 (c 0.5, CH3OH); HNMR (DMSO-d6, 400 MHz,) δ
D
7.65 (1H, s, NH), 7.35 (s, 1H, H-7), 7.31(1H, s, H-10), 6.35 (1H,
m, H-1), 6.06-6.05 (2H, m, -OCH2O-), 5.71 (1H, d, J ) 6.5
Hz, OH), 4.26-4.22 (2H, m, H-4a, H-2), 3.93-3.79 (2H, m, H-3,
H-4); 13C NMR (DMSO-d6, 125 MHz) 179.5 (C, C-6) 161.4 (C),
151.2 (C), 147.7 (C), 128.9 (C), 126.2 (CH, C-1), 120.7 (C), 106.1-
(C), 101.8 (CH2, CH, -OCH2O-, C-7), 75.6 (CH, C-4), 74.8 (CH,
C-3), 70.3 (CH, C-2), 54.04 (CH, C-4a); 31P (DMSO-d6, 162 MHz)
δ 5.23; HRESI m/z 352.0202 [M - Na]- (calcd for C14H11NOP-
352.0222 [M - Na]-).
needles: mp 238-240 °C; [R]23 +151° (c 1.04, CH2Cl2) [lit.9c
D
244-246 °C, [R]27D +219° (c 1.0, CHCl3)]; 1H NMR(CDCl3, 300
MHz) δ 7.50 (1H, s, H-7), 7.01 (1H, s, NH), 6.99 (1H, s, H-10),
6.10 (1H, m, H-1), 6.06-6.04 (2H, m, -OCH2O-), 5.46 (1H, m,
H-3), 5.34 (1H, m, H-2), 5.26-5.23 (1H, dd, J ) 9.3, 2.1 Hz,
H-4) 4.66 (1H, bd, J ) 9.3 Hz, H-4a) 2.15 (3H, s, -OCOCH3),
2.1 (3H, s, -OCOCH3), 2.09 (3H, s, -OCOCH3); 13CNMR-
(CDCl3, 300 MHz) δ 169.9 (C, OCOCH3), 169.2 (2C, OCOCH3),
163.8 (C, C-6), 151.3 (C), 148.7 (C), 133.6 (C), 129.8 (C), 121.8
(C), 116.7 (CH, C-1), 107.0 (CH, C-7), 102.9 (CH, C-10), 101.6
(CH2, -OCH2O-), 70.7 (CH, C-4), 68.0 (CH, C-2), 67.7 (CH,
C-3), 49.7 (CH, C-4a), 20.5 (CH3, -OCOCH3), 20.4 (CH3,
-OCOCH3), 20.2 (CH3, -OCOCH3); HRAPCI+ m/z 418.1134
[M + 1]+ (calcd for C20H20NO9 418.1138 [M + 1]+).
General Procedure for the Preparation of 7-Deoxy-
narcistatin Prodrugs 5a and 5b. Sodium 7-deoxynarcistatin
(5, 52 mg) was dissolved in H2O (1 mL) and the solution passed
through a column of Dowex 50WX8-400, bearing the respective
cation. The UV-active fractions were combined and freeze-dried
to give the corresponding narcistatin salt as a white solid.
Lithium 7-deoxynarcistatin (5a): 34 mg, mp 250 °C (dec);
1H NMR (DMSO-d6, 300 MHz) δ 7.65 (1H, s, NH), 7.37 (1H, s,
H-7), 7.32 (1H, s, H-10), 6.38 (1H, m, H-1), 6.08-6.07 (2H, m,
-OCH2O-), 5.72 (1H, d, J ) 6 Hz, OH), 4.30-4.25 (2H, m,
H-4a, H-2), 3.91-3.78 (2H, m, H-3, H-4).
Recrystallization of the precipitate (3b) from CH2Cl2-CH3-
OH gave colorless needles: mp 208 °C, [R]23 +103° (c 0.53
Potassium 7-deoxynarcistatin (5b): 43 mg, mp 230-235
D
CH2Cl2) [lit.15b mp 148-149 °C]; 1HNMR (CDCl3, 300 MHz) δ
7.55 (1H, s, H-7), 6.72 (1H, s, 1H), 6.18 (1H, s, NH), 6.03 (2H,
s, -OCH2O-), 5.43 (1H, t, J ) 3 Hz, H-3), 5.22-5.18 (2H, m,
H-3, H-4), 3.79 (1H, dd, J ) 12.75, 10.95, H-4a), 3.18 (1H, m,
°C (dec); H NMR (DMSO-d6, 300 MHz) δ 7.67 (1H, s, NH),
1
7.37 (1H, s, H-7), 7.32 (1H, s, H-10), 6.37 (1H, m, H-1), 6.08-
6.07 (2H, m, -OCH2O-), 5.70 (1H, m, OH), 4.24 (2H, bm,
H-4a, H-2), 3.91-3.78 (2H, m, H-3, H-4).

210 Journal of Natural Products, 2005, Vol. 68, No. 2
Pettit and Melody
7-Deoxy-trans-dihydronarciclasine (3a). The saponifica-
tion of 2,3,4-triacetoxy-7-deoxy-trans-dihydronarciclasine (3b,
0.14 g) was performed in 9:1 aqueous MeOH-K2CO3 (0.003
g) and was conducted as described above for obtaining alcohol
1b to yield triol 3a as a colorless solid: 89 mg (91% yield); mp
H-1ax); 13C NMR (DMSO-d6, 100 MHz) δ 169.5 (C-6), 163.9
(C), 152.1 (C), 145.5 (C), 137.6 (C), 106.9 (C), 101.8 (CH2,
-OCH2O-), 96.7 (CH, C-10), 74.9 (3CH, C-2, C-3, C-4), 56.1
(CH, C-4a), 31.5 (CH, C-10b), 28.7 (CH2,C-1); 31P NMR (DMSO-
d6, 162 MHz) δ 13.36; HRESI m/z 370.0314 [M - Na]- (calcd
for C14H13NO9P- 370.0328 [M - Na]-).
1
>300 °C (dec) [lit.10a mp 303-304 °C]; H NMR (CDCl3, 300
MHz) δ 7.28 (1H, s, NH), 6.91 (2H, m, H-7, H-10), 6.05 (2H,
m, -OCH2O-), 4.95-4.90 (2H, m, OH), 4.78 (1H, s, OH), 3.86
(1H, m, H-4a), 3.69 (2H, m, H-3, H-4), 3.3 (1H, m, H-2), 2.86
General Procedure for the Preparation of trans-
Dihydronarcistatin Prodrugs 7a and 7b. Sodium trans-
dihydronarcistatin (7, 0.010 g) was dissolved in water (1 mL)
and the solution passed through a Dowex 50WX8-200 column,
bearing the respective cation. The UV-active fractions were
combined and freeze-dried to give the corresponding trans-
dihydronarcistatin salt as a white solid.
Lithium trans-dihydronarcistatin (7a): 8 mg, mp 275
°C (dec); 1H NMR (DMSO-d6, 300 MHz) δ 13.17 (1H, s, OH-7),
8.41 (1H, s, NH), 6.52 (1H, s, H-10), 6.04-6.02 (2H, m,
-OCH2O-), 5.23 (1H, s, OH), 4.14-4.02 (3H, m, H-2, 3, 4),
3.34-3.57 (1H, m, H-4a), 2.88-2.79 (1H, m, H-10b), 2.28-2.24
(1H, m, H-1eq), 1.52-1.45 (1H, m, H-1ax).
Potassium trans-dihydronarcistatin (7b): 7.1 mg, mp
230-235 °C (dec); 1H NMR (DMSO-d6, 300 MHz) δ 13.16 (1H,
s, OH-7), 8.41 (1H, s, NH), 6.51 (1H, s, H-10), 6.04-6.02 (2H,
m, -OCH2O-), 5.19 (1H, s, OH-2), 4.09-4.04 (3H, m, H-2, 3,
4), 3.57 (1H, m, H-4a), 2.82 (1H, m, H-10b), 2.28-2.22 (1H,
m, H-1eq), 1.53-1.43 (1H, m, H-1ax).
Preparation of Sodium Narcistatin (8). Synthesis of 3,4-
cyclic phosphate 8 from narciclasine (1a) (0.113 g, 0.368 mmol)
was carried out in pyridine (4 mL) using tetrabutylammonium
dihydrogen phosphate (0.075 g, 0.22 mmol) and dicyclohexy-
lcarbodiimide (0.4 g, 1.94 mmol) with additional amounts of
tetrabutylammonium dihydrogen phosphate (0.185 g) and
dicyclohexylcarbodiimide (0.4 g) added after the first 24 h
stirring at 80 °C. The reaction was stirred for 96 h, cooled,
and filtered to remove precipitated DCU. Water (100 mL) was
added and the mixture refiltered to remove any residual DCU.
The mother liquor was concentrated to minimum volume. The
aqueous fraction was eluted through an ion-exchange column
(sodium form). The UV-active fractions were combined and
lypholized to yield the phosphate 8 as a cream solid (88%
yield). Analysis (1H NMR, 13C NMR, 31P NMR) showed 8 to be
identical with an authentic sample.13a
(1H, m, H-10b), 2.12 (1H, m, H-1eq), 1.64 (1H, m, H-1ax); 13
C
NMR (DMSO-d6, 75 MHz) δ 164.9 (C-6), 151.3 (C), 138.7 (2C)
123.9 (C), 107.6 (CH, C-7), 104.9 (CH, C-10), 102.2 (CH2,
-OCH2O-), 72.3 (CH, C-4), 70.4 (CH, C-3), 69.3 (CH, C-2), 55.8
(CH, C-4a), 34.9 (CH, C-10b), 29.9 (CH, C-1); HRFAB+ m/z
294.0967 [M + H]+ (calcd for C14H15NO6 294.0978 [M + H]+).
Sodium 7-Deoxy-trans-dihydronarcistatin (6). The con-
version of 7-deoxy-trans-dihydronarciclasine (3a, 0.15 g, 0.51
mmol) to narcistatin 6 in pyridine (6 mL) with tetrabutylam-
monium dihydrogen phosphate (0.21 g, 0.62 mmol, 1.2 equiv)
and dicyclohexylcarbodiimide (0.54 g, 2.62 mmol, 5.13 equiv)
was conducted and the phosphate isolated as summarized for
synthesis of narcistatin (5) including the additional tetrabu-
tylammonium dihydrogen phosphate (0.21 g, 1.2 equiv) and
DCCI (0.54 g) (cf. above). The aqueous extract of product was
subjected to a Dowex 50WX8-400 ion-exchange column (so-
dium form), and the UV fluorescing fractions were combined
and lyophilized as noted above (cf. 5). A solution of the sodium
salt was prepared in MeOH (15 mL with heating), the insoluble
material was collected, and the filtrate was concentrated to
yield 0.124 g, 65%; mp 297 °C (dec); [R]23D -19° (c 0.35, H2O);
1H NMR (DMSO-d6, 400 MHz6) δ 7.53 (1H, s, NH), 7.27 (1H,
s, H-7), 6.93 (1H, s, H-10), 6.04-6.03 (2H, m, -OCH2O-), 5.22
(1H, bs, OH), 4.12-4.09 (1H, m, H-3)), 4.07-3.98 (2H, m, H-2,
H-4), 3.54 (1H, dd, J ) 13, 8.6 Hz, H-4a), 2.84 (1H, m, H-10b),
2.27 (1H, m, H-1eq), 1.47 (1H, m, H-1ax); 13C NMR (DMSO-
d6, 100 MHz) δ 164.7 (C, C-6), 151.2 (C), 146.7 (C), 139.7 (C),
124.2 (C), 107.6 (CH, C-7), 105.2 (CH, C-10), 102.2 (CH2,
-OCH2O-), 75.9 (CH, C-4), 75.7 (CH, C-3), 60.6 (CH, C-2),
56.9 (CH, C-4a), 32.7 (CH, C-10b), 29.5 (CH2, C-1); 31P NMR
(162 MHz, DMSO-d6) δ 13.45; HRESI m/z 354.5294 [M - Na]-
(calcd for C14H13NO8P- 354.0379 [M - Na]-).
General Procedure for the Preparation of 7-Deoxy-
trans-dihydronarcistatin Prodrugs 6a and 6b. Sodium
7-deoxy-trans-dihydronarcistatin (6, 30 mg) was dissolved in
H2O (1 mL) and the solution passed through a column of
Dowex 50WX8-400, bearing the respective cation. The UV-
active fractions were combined and freeze-dried to give the
corresponding narcistatin salt as a white solid.
Lithium 7-deoxy-trans-dihydronarcistatin (6a): 25.4
mg, mp 253 °C (dec); 1H NMR (DMSO-d6, 300 MHz) δ 7.57
(1H, s, NH), 7.28 (1H, s, H-7), 6.96 (1H, s, H-10), 6.06-6.05
(2H, m, -OCH2O-), 5.19 (1H, m, OH), 4.10-4.00 (3H, m, H-4,
H-3, H-2), 3.57 (1H, dd, J ) 8.1, 12.9 Hz, H-4a), 2.86 (1H, m,
H-10b), 2.28 (1H, m, H-1eq), 1.50 (1H, m, H-1ax).
Acknowledgment. We are pleased to acknowledge finan-
cial support provided by Outstanding Investigator Grant
CA44344-03-12 and RO1 CA90441-01-03 awarded by the
Division of Cancer Treatment and Diagnosis, National Cancer
Institute, DHHS; the Arizona Disease Control Research Com-
mission; the Robert B. Dalton Endowment Fund; Dr. Alec D.
Keith; the Caitlin Robb Foundation; Gary L. and Diane R.
Tooker; Polly J. Trautman; John C. Budzinski; the Eagles Art
Ehrmann Cancer Fund; and the Ladies Auxiliary to the
Veterans of Foreign Wars. Other helpful assistance was
provided by Drs. D. L. Doubek, L. C. Garner, F. Hogan, J. C.
Knight, V. J. R. V. Mukku, J. Chapuis, and J. M. Schmidt; G.
R. Pettit III, L. A. Richert, R. G. Thornburgh, and L. Williams;
and the Washington University Mass Spectrometry Labora-
tory, a NIH Research Resource (Grant P41RR0954).
Potassium 7-deoxy-trans-dihydronarcistatin (6b): 23.2
mg, mp 287 °C (dec); 1H NMR (DMSO-d6, 300 MHz) δ 7.56
(1H, s, NH), 7.28 (1H, s, H-7), 6.96 (1H, s, H-10), 6.06 (2H, s,
-OCH2O-), 5.17 (1H, m, OH), 4.07-3.95 (3H, m, H-3, H-2,
H-4), 3.59-3.52 (1H, m, H-4a), 2.84 (1H, m, H-10b), 2.28 (1H,
m, H-1eq), 1.48 (1H, m, H-1ax).
Sodium trans-dihydronarcistatin (7). Synthesis of 3,4-
cyclic phosphate 7 from trans-dihydronarciclasine18,19 (4, 57
mg, 0.184 mmol) was accomplished in pyridine (2 mL) employ-
ing tetrabutylammonium dihydrogen phosphate (60 mg, 0.176
mmol, and 60 mg for the delayed addition) and dicyclohexyl-
carbodiimide (0.18 g, 0.87 mmol, and 0.18 g for the second
addition) as described for the preparation of phosphate 5 (see
above). The aqueous fraction eluted from the ion-exchange
column (Dowex 50WX8-400, sodium form) provided sodium
trans-dihydronarcistatin (7) as a colorless solid (86 mg, 94%
yield): mp > 300 °C, [R]23D -32° (c 0.3, H2O); 1H NMR (DMSO-
d6, 400 MHz) δ 13.16 (1H, s, OH-7), 8.40 (1H, s, NH), 6.52
(1H, s, H-7), 6.03 (2H, m, -OCH2O-), 5.26 (1H, bs, OH), 4.14-
4.03 (3H, m, H-3,H-4, H-2), 3.59 (1H, dd, J ) 13.6, 8.8 Hz,
H-4a), 2.84 (1H, m, H-10b), 2.26 (1H, m, H-1eq), 1.50 (1H, m,
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NP0304518