Synthesis and structure-activity relationships study of novel anti-tumor carbamate anhydrovinblastine analogues

Article abstract of DOI:10.1016/j.bmc.2007.05.045

A series of 3-demethoxycarbonyl-3-carbamate methyl anhydrovinblastine derivatives (compounds 8b-32b) were designed, synthesized, and evaluated for their inhibition activities against human non-small cell lung cancer cell line (A549) and a human cervix epi

Full text of DOI:10.1016/j.bmc.2007.05.045

Bioorganic & Medicinal Chemistry 15 (2007) 5061–5075
Synthesis and structure–activity relationships study of novel
anti-tumor carbamate anhydrovinblastine analogues
Yong Shao,a Hong Ding,b Weidong Tang,b Liguang Loub,* and Lihong Hua,*
aShanghai Research Center for Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia
Medica, Chinese Academy of Sciences, Shanghai 201203, PR China
bDepartment of Pharmacology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR China
Received 26 March 2007; revised 11 May 2007; accepted 17 May 2007
Available online 23 May 2007
Abstract—A series of 3-demethoxycarbonyl-3-carbamate methyl anhydrovinblastine derivatives (compounds 8b–32b) were
designed, synthesized, and evaluated for their inhibition activities against human non-small cell lung cancer cell line (A549) and
a human cervix epithelial adenocarcinoma cell line (HeLa). The structure–activity relationships of this new series are described
in this paper. Cytotoxicity data revealed that the size of substituents and substitution position had important influence on cytotoxic
activity. On two cell lines, compounds (8b and 30b) had more potent cytotoxic activity than the lead compound (1e, AVLB). The
preliminary antitumor studies of 8b in vivo showed that it might be promising for the development of new antitumor agents.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
shown to decrease toxicity to inhibit the growth of
lymphomas and certain solid tumors, specifically cervi-
cal, lung, breast, and colon cancer.8 In order to inves-
tigate structural requirement of cytotoxic activity of
AVLB, we were interested in exploiting the 22-posi-
tion in the vindoline moiety of anhydrovinblastine as
part of an extended SAR study. In this report, a
new series of 3-demethoxycarbonyl-3-carbamate
methyl anhydrovinblastine derivatives were synthesized
and tested for cytotoxic activity in vitro against A549
and HeLa cell lines.
Vinblastine (1a) and vincristine (1b) have been recog-
nized as clinically important antitumor agents because
of their strong antineoplastic activity against a wide
spectrum of human tumors for more than 40 years
(Fig. 1).1 These two alkaloids, although structurally
almost identical, differ markedly in the type of tumors
that they affect and in their toxicological properties.2
Therefore, it is desirable to identify structural modifi-
cation that would yield more effective and less toxic
analogues. Most of research has mainly focused on
the semi-synthesis or total synthesis of vinblastine ana-
logues from the two parts of the dimeric structure, the
vindoline and velbenamine portions, by carbon skele-
ton modification and functional group transforma-
tion.3,4 To date, these efforts have led to two
approved drugs, vindesine (1c)5 and vinorelbine (1d,
NVB)6, and more than a dozen candidates in clinical
evaluation, such as anhydrovinblastine (1e, AVLB)7.
Compared with vincristine and NVB, AVLB has been
R4
N
n
R5
H
7
6
N
N
H
Et
OH
3
22
R2
H3CO2C
2
N
R
H3CO
OR3
H
1O
Vindoline moiety
R1 R2
R3
R4 R5
n
1
1
1
0
1
1a Me OMe COMe OH
1b CHO OMe COMe OH
H
H
H
Keywords: Antitumor; Anhydrovinblastine derivatives; Carbamate;
Cytotoxicity.
1c Me NH2 OH
1d Me OMe COMe
1e Me OMe COMe
OH
=
=
*
Corresponding authors. Tel./fax: +86 21 50806056 (L.L.); tel./fax:
+86 21 50272221 (L.H.); e-mail addresses: lglou@mail.shcnc.ac.cn;
simmhulh@mail.shcnc.ac.cn
Figure 1. Vinca alkaloids structure.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.05.045

5062
Y. Shao et al. / Bioorg. Med. Chem. 15 (2007) 5061–5075
2. Chemistry
(80%), which were subsequently coupled with 3 to pro-
duce the carbamate anhydrovinblastine derivatives
(8b–30b). Chloroformates could be obtained from com-
mercial suppliers or be synthesized by different alcohols
with bis(trichloromethyl)carbonate in the presence of
DIPEA in DCM.12 Synthesis of inner carbamate com-
pound 31b and reverse carbamate compound 32b was
also achieved for better complement of the SARs
according to Scheme 2. Compound 21a underwent intra-
molecular attack reaction with sodium hydride to afford
31a, and then coupled with 3 to produce compound 31b.
For synthesis of compound 32a, 32 was first prepared by
isobutyl amine and carbonyldiimidazole (CDI) in DCM
with good yield.13 Subsequently, the triol 4 was treated
with 32 in the presence of sodium hydride in THF and
then acetylated to yield compound 32a, which was cou-
pled with 3 to furnish the compound 32b.
Most of semi-synthesized vinblastine derivatives reported
previously were prepared by functional group transfor-
mation from vinblastine. Due to its complex structural
features and chemical instability, reactions used in func-
tional group transformations usually needed mild condi-
tions. Under these conditions our designed carbamate
analogues could not be afforded by anhydrovinblastine
as starting material. As we know, coupling of the two bio-
synthetical precursor monomers, vindoline (2) and catha-
ranthine (3), can form 30,40-anhydrovinblastine by the
modified Polonovski-Potier reaction.9 Vindoline10, the
major alkaloid in whole plant of Catharanthus roseus,
was easier to be obtained and modified than the dimer
vinca alkaloids. Thus, our synthetical strategy was to first
prepare the amine (7) and its carbamate derivatives (8a–
32a) from vindoline (2), and then couple with 3 to prepare
the targeted derivatives (8b–32b).
3. Result and discussion
As shown in Scheme 1, the important intermediate
amine (7) was prepared in following four steps. Reduc-
tion of 2 by LiAlH4 could provide the triol 4 (98%),
which was treated with 4-toluene sulfonyl chloride in
THF including 50% sodium hydroxide solution at
80 ꢁC to result in the formation of 3b,22-epoxide (5) in
80% yield. The ring-opening reaction of the epoxide
(5) afforded 6 in excellent yield (91%) with azide in the
presence of NH4Cl, which was easy to be purified.11
Treatment of 6 with LiAlH4 at 0 ꢁC provided 7 in 70%
total yield from vindoline. Then 7 was treated with dif-
ferent chloroformats in the presence of diisopropylethyl-
amine (DIPEA) in dichloromethane (DCM) and then
acetylated for 4-OH to furnish compounds 8a–30a
Total 25 dimeric compounds (8b–32b) were tested for
their cytotoxicity against human non-small cell lung
cancer cell line (A549) and a human cervix epithelial
adenocarcinoma cell line (HeLa) by sulforhodamine B
(SRB) assay14 employing AVLB as positive control.
The IC50 values for the inhibition of proliferation are
shown in Tables 1 and 2. The details for bioassay proce-
dures are described in Section 5.
The methyl carbamate derivative (8b) initially synthe-
sized showed more potent cytotoxic activity (IC50 values
on A549 and HeLa cell lines are 38 and 9 nM, respec-
tively) than positive control AVLB (49 nM and
10
N
N
N
7
6
14
19
b
a
Et
OH Et
OH
OH Et
O
O
N
OH
N
O
c
N
OAc
COOMe
3
17
H
H
O
H
22
OH
4
5
2 Vindoline
N
N
N
e, f
d
OH Et
OAc
OH Et
OH Et
OH
O
N
O
N
OH
O
N
H
H
H
NH
NH2
N3
8a ~ 30a
7
6
O
OR
g
N
N
H
N
N
H
N
H
MeO
O
H3CO2C
OH Et
OAc
H3CO
3 Catharanthine
N
H
NH
OR
8b ~ 30b, table 1
O
Scheme 1. Reagents and conditions: (a) LiAlH4, THF, 0 ꢁC–rt, 4 h, 98%; (b) i—THF, 50%NaOH–H2O, Bu4N+Iꢀ, 50 ꢁC, 30 min; ii—4-toluene
sulfonyl chloride, 80 ꢁC, 3 h, 80%; (c) NaN3, CH3OH/H2O (8:1), 90 ꢁC, 24 h, 91%; (d) LiAlH4, THF, 0 ꢁC–rt, 4 h, 98%; (e) ClCOOR, DIPEA, DCM;
(f) Ac2O, pyridine; (g) i—FeCl3, HCl–H2O buffer (pH 2), 8 h; ii—NaBH4, NH4OH, 0 ꢁC, 15 min.

Y. Shao et al. / Bioorg. Med. Chem. 15 (2007) 5061–5075
5063
N
N
N
3, b
a
OH Et
Et
Et
O
N
O
N
OAc
OAc
O
H3CO
N
OAc
H
H
O
H
NH
N
H
O
N
O
H
O
O
O
21a
31a
31b
N
N
OH Et
OH Et
3, b
32, d
H3CO
N
OAc
N
OAc
4
H
H
e
O
O
O
N
H
O
N
H
32a
32b
O
N
N
H
c
NH2
N
32
Scheme 2. Reagents and conditions: (a) NaH, THF, 0 ꢁC–rt, 2 h, 40%; (b) i—FeCl3, HCl–H2O buffer (pH 2), 8 h; ii—NaBH4, NH4OH, 0 ꢁC, 15 min;
(c) CDI, DCM, 0 ꢁC–rt, 24 h; (d) NaH, THF, 0 ꢁC–rt, 4 h; (e) Ac2O, pyridine.
26 nM). These results indicated that it was suitable for
improving the cytotoxicity to introduce the carbamate
group at C22 of vindoline moiety. So, our present work
focused on exploring variants of the substituted alkyl
and aromatic groups in 8b and studying their effects
on cytotoxicity. From Table 1, we observed a significant
loss of cytotoxicity against HeLa cell with increasing the
size of alkyl groups by one additional carbon (8b, 9b,
10b, 11b, and 15b). These results also could be obtained
from the aliphatic ring substitution (17b, 18b, and 19b).
Reverse carbamate was found to be much less active
(32b vs 12b), which showed the C22–NH was favorable
to its cytotoxic activity. 2-Methoxyl ethyl carbamate
(13b) had comparable activity to the ethyl carbamate
(9b), while 2-bromoethyl group (14b) was much less ac-
tive (IC50 = 14 nM for 13b vs 16 nM for 9b vs 30 nM for
14b). Cyclopropylmethyl carbamate (16b) also demon-
strated potent inhibitory activity (IC50 = 13 nM), which
had more activity than isobutyl group (12b,
IC50 = 67 nM). The spiro carbamate (31b) showed low
activities (IC50 = 387 nM), indicating the alkyl substitu-
ents are favorable to their cytotoxicities. Similar SAR
results of alkyl carbamates also could be found aganist
the A549 cell line. Overall, the size of alkyl substituents
had more effect on A549 than on HeLa cell line. Small
alkyl carbamate analogues, such as 8b, 9b, 10b, 13b,
16b, and 17b, had more than or equal activity to AVLB
against HeLa cell line, while only 8b had more activity
against A549.
estingly, we have found 23b (54 nM) had a significant in-
crease of cytotoxicity against HeLa cell with benzyl
group instead of phenyl group. Subsequently, we further
investigated the impact of different substituted benzyl
groups on their cytotoxicities. Introduction of elec-
tron-donating (OMe) or electron-withdrawing groups
(Cl and NO2) at the 4-position of benzyl group afforded
compounds (25b, 28b, and 30b) with a 3-fold increase in
cytotoxic activity against HeLa cell line than AVLB,
while 2-methoxyl (24b), 3-chloro (27b), and 2-nitro ben-
zyl carbamates (29b) showed slightly lower activities.
Piperonyl (26b) was only slightly less cytotoxic than sin-
gle methoxyl substituent at 4-position (25b). However,
2-methoxyl (24b) and 3-chloro benzyl carbamate (27b)
were 4-fold less active against A549 cell line than AVLB,
4-methoxyl (25b) and 4-chloro (28b) also showed slightly
lower activity. Only 4-nitro benzyl carbamate (30b,
IC50 = 38 nM) showed potency increase as methyl car-
bamate (8b) than AVLB. In general, all aromatic carba-
mate analogues were less sensitive against A549 than
HeLa cell line. The same SAR results were to be found
that 4-substituted benzyls were beneficial to their cyto-
toxicities, and benzyl carbamates were more potent than
phenyl carbamates.
Compound 8b, which was both potent on two cell lines
and easily prepared, was chosen to further evaluate anti-
tumor efficacy in vivo. The antitumor activity of 8b
in vivo was evaluated at doses of 1.5, 3, and 6 mg/kg
in nude mice bearing A549 xenografts as the animal
model in comparison with compound 1d. Compound
8b demonstrated a dose-dependent inhibition of A549
tumor and superior tumor inhibitory activity with
68.5% inhibition at a dose of 6 mg/kg, which showed
comparable effect to the clinical drug vinorelbine (1d)
at a dose of 10 mg/kg. The detailed experimental data
are shown in Table 3.
In our further study, carbamates substituted with aro-
matic groups were synthesized and tested. Compounds
20b, 21b, and 22b also showed low activities against
HeLa cell line for their steric effects. (Table 2) Among
the three analogues, 4-methoxy-substituted phenyl
group was beneficial to its cytotoxic activity (IC50 values
for 22b and 20b are 49 and 199 nM, respectively). Inter-

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Y. Shao et al. / Bioorg. Med. Chem. 15 (2007) 5061–5075
Table 2. Cytotoxic activity of aromatic substituted analogues
Table 1. Cytotoxic activity of aliphatic substituted analogues
Compounda
AVLB
R
IC50 (lM)
HeLa
Compounda
R
IC50 (lM)
HeLa
A549
A549
0.049 0.005 0.026 0.002
0.038 0.003 0.009 0.001
0.051 0.004 0.016 0.001
0.162 0.011 0.027 0.003
0.604 0.011 0.096 0.009
0.066 0.005 0.067 0.008
0.065 0.008 0.014 0.001
0.131 0.017 0.030 0.003
0.426 0.036 0.184 0.014
0.061 0.004 0.013 0.002
0.121 0.007 0.023 0.002
0.177 0.023 0.132 0.009
0.381 0.037 0.250 0.026
20b
21b
22b
23b
24b
25b
26b
27b
28b
0.110 0.010 0.199 0.018
0.609 0.068 0.148 0.005
0.365 0.032 0.049 0.005
0.057 0.008 0.054 0.002
0.200 0.014 0.069 0.003
0.067 0.009 0.008 0.001
0.075 0.007 0.017 0.002
0.203 0.018 0.039 0.005
0.089 0.011 0.006 0.001
O
O
8b
O
O
OMe
OMe
9b
O
O
10b
11b
12b
13b
14b
15b
16b
17b
18b
19b
O
OMe
O
O
O
O
OMe
O
O
O
O
O
O
Br
Cl
O
O
O
O
Cl
NO2
29b
30b
0.061 0.009 0.066 0.007
0.038 0.004 0.010 0.001
O
O
O
O
O
NO2
a Ditartrate of all compounds was used in bioassays.
cytotoxic activities in two tumor cell lines. The prelimin-
ary antitumor studies of 8b in vivo showed that it might
be promising for the development of new antitumor
agents.
NH
O
31b
32b
0.631 0.051 0.387 0.035
0.485 0.020 0.186 0.014
O
N
5. Experimental
H
a Ditartrate of all compounds was used in bioassays.
5.1. Materials and methods
Unless otherwise mentioned, all chemicals and materials
were used as received from commercial suppliers with-
out further purification. Tetrahydrofuran (THF) was
distilled from sodium/benzophenone under nitrogen.
Dichloromethane was distilled from calcium hydride.
Thin layer chromatography (TLC) plates (silica gel 60
GF, with glass support) from Yantai jiangyou company
were used for monitoring progress of a reaction and
visualized with 254 nm UV light and/or spray by a
10% solution of ceric ammonium sulfate (CAS) in phos-
phoric acid. NMR spectra were recorded on a Varian
Mercury-VX300 Fourier transform spectrometer or a
Bruker AM-400 spectrometer. The chemical shifts are
4. Conclusion
A series of carbamate anhydrovinblastine derivatives
(8b–32b) were designed and synthesized. All the com-
pounds were evaluated for their growth inhibition activ-
ities against two tumor cell lines (A549 and HeLa). The
SAR information collected so far suggested that the size
and position of the carbamate substituents at the 22-po-
sition were both important factors for cytotoxic activity
in vitro. Compounds 8b and 30b maintained potent

Y. Shao et al. / Bioorg. Med. Chem. 15 (2007) 5061–5075
5065
Table 3. In vivo anti-tumor efficacy of compound 8ba against non-small cell lung cancer A549 xenografts
Group
Dose (mg/kg)
Mice (n)
Body weight (g)
Tumor volume (mm3)
SD
Relative TV (X SD)
Inhibition (%)
X
Initial/end
Initial/end
Initial
end
Control
8b
8b
12/12
6/6
6/6
18.8/21.4
19.2/21.3
19.3/20.4
19.1/18.8
19.0/16.4
214 48
256 41
228 68
241 30
218 54
2194 868
2080 542
1545 410
808 336
550 206
11.06 5.49
8.18 2.20
6.96 1.79
3.48 1.83
2.66 1.18
1.5
3
26.0
37.1*
68.5*
75.9*
8b
1d
6
10
6/6
6/6
a The in vivo experiment was carried out in the nude mice bearing A549 xenografts and the compounds were intravenously given. Ditartrate of all
compounds was used in bioassays.
* P < 0.01 versus control group.
reported in ppm using the d 7.26 signal of CDCl3 (1H
NMR) and the d 77.23 signal of CDCl3 (13C NMR) as
internal standards. IR spectra were recorded on a Per-
kin-Elmer 577 spectrometer. EI-MS was obtained on a
SHIMADZU GCMS-QP5050A spectrometer. ESI-MS
was run on a Bruker Esquire 3000 plus spectrometer
in MeOH.
400 MHz) d: 6.84 (d, J = 8.0 Hz, 1H), 6.10 (dd, J = 8.0,
2.4 Hz, 1H), 6.03 (dd, J = 10.0, 4.8 Hz, 1H), 5.90 (d,
J = 2.4 Hz, 1H), 5.48 (d, J = 10.0 Hz, 1H), 4.07 (s, 1H),
3.78 (s, 3H), 3.54 (dd, J = 16.4, 4.8 Hz, 1H), 3.34–3.30
(m, 1H), 3.01 (s, 1H), 2.97 (s, 1H), 2.94 (s, 3H), 2.88 (dt,
J = 16.4, 2.0 Hz, 1H), 2.57 (d, J = 4.0 Hz, 1H), 2.48 (d,
J = 4.0 Hz, 1H), 2.42–2.34 (m, 2H), 1.67–1.63 (m, 1H),
1.41–1.35 (m, 1H), 1.12–1.07 (m, 1H), 0.79 (t,
J = 7.2 Hz, 3H); 13C NMR (CDCl3, 75 MHz) d: 161.1,
151.3, 130.5, 127.1 (2C), 121.2, 100.4, 92.3, 78.6, 68.7,
64.6, 63.7, 55.3, 54.2, 53.3, 51.6, 50.8, 45.4, 40.4, 33.6,
27.2, 8.4. EIMS (m/z) 368 (M+), 339, 297, 188, 174, 121,
107, 93.
5.1.1. 3,4,22-Triol-3-demethoxycarbonyl-3-hydroxyl-
methyl-4-deacetyl-vindoline (4). To a suspension of
LiAlH4 (114 mg, 3.00 mmol) in dry THF (10 mL) at
0 ꢁC, a solution of vindoline (486 mg, 1.00 mmol) in
dry THF (10 mL) was added slowly. The reaction mix-
ture was stirred at room temperature for 4 h and then
quenched cautiously by the subsequent addition of
water (114 lL), 15% NaOH (aq) (114 lL), and water
(342 lL). The resulting suspension was warmed to room
temperature and stirred for 10 min. The suspension was
filtered through a fritted funnel and the filtrate was con-
5.1.3. 3-Demethoxycarbonyl-3-azidomethyl-4-deacetyl-vin-
doline (6). A solution of compound 5 (8 mmol, 2.95 g) in
a 8:1 MeOH/H2O mixture (72 mL) was treated with
NaN3 (2.6 g, 40 mmol) and NH4Cl (0.936 g, 17.6 mmol),
and the reaction mixture was stirred at 80 ꢁC for 20 h.
After cooling EtOAc (100 mL)and H2O (100 mL) were
added. The aqueous layer was additionally extracted with
EtOAc (2· 30 mL) and the combined organic layer was
washed with brine (100 mL) and dried (Na2SO4). Removal
of the solvents was followed by column chromatography
centrated in vacuo to give crude triol. The product was
20
used in the next step without purification. ½aꢁ þ 45ꢂ (c
D
0.20, CHCl3), 1H NMR (CDCl3, 400 MHz) d: 8.73 (br s,
1 H), 6.82 (d, J = 8.0 Hz, 1H), 6.30 (dd, J = 8.0, 2.4 Hz,
1H), 6.12 (d, J = 2.4 Hz, 1H), 5.88 (dd, J = 10.0, 4.8 Hz,
1H), 5.60 (d, J = 10.0 Hz, 1H), 3.93 (d, J = 11.2 Hz, 1H),
3.78 (s, 3H), 3.72 (d, J = 11.2 Hz, 1H), 3.52 (s, 2H), 3.45
(dd, J = 15.6, 4.4 Hz, 1H), 3.35 (td, J = 9.2, 4.4 Hz, 1H),
3.00 (s, 3H), 2.83 (dt, J = 15.6, 2.0 Hz, 1H), 2.57 (s, 1H),
2.53–2.50 (m, 2H), 2.25–2.16 (m, 3H), 1.36–1.31 (m,
1H), 0.94–0.88 (m, 1H), 0.62 (t, J = 7.6 Hz, 3H); 13C
NMR (CDCl3, 75 MHz) d: 160.9, 154.6, 130.8, 126.5,
124.3, 122.9, 104.6, 96.3, 81.0, 77.4, 75.2, 68.3, 65.7,
55.4, 51.8, 51.7, 51.3, 44.8, 43.5, 40.4, 32.5, 7.9. EIMS
(m/z) 386 (M+), 368, 355, 297, 212, 188, 174, 162, 135,
93.
on silica gel (8:1, hexanes/acetone) to afford the compound
20
D
CHCl3), IR (KBr): 3405, 2964, 2102, 1616, 1500, 1463,
6 as a white solid in 91% (2.99 g) yield. ½aꢁ ꢀ 69ꢂ (c 0.18,
.
1278, 1222, 1164, 1084 cmꢀ1 1H NMR (CDCl3,
400 MHz) d: 8.68 (br s, 1H), 6.87 (d, J = 8.4 Hz, 1H),
6.30 (dd, J = 8.4, 2.4 Hz, 1H), 6.09 (d, J = 2.4 Hz, 1H),
5.88 (dd, J = 10.4, 4.8 Hz, 1H), 5.58 (d, J = 10.4 Hz, 1H),
3.85 (d, J = 11.2 Hz, 1H), 3.78 (s, 3H), 3.61 (s, 1H), 3.46
(dd, J = 16.0, 4.8 Hz, 1H), 3.42–3.37 (m, 2H), 3.20 (d,
J = 11.2 Hz, 1H), 2.93 (s, 3H), 2.81 (dt, J = 16.0, 2.0 Hz,
1H), 2.51 (s, 1H), 2.48–2.43 (m, 1H), 2.29–2.23 (m, 2H),
1.92 (d, J = 12.4 Hz, 1H), 1.29–1.23 (m, 1H), 0.89–0.83
(m, 1H), 0.58 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3,
75 MHz) d: 161.0, 154.8, 130.2, 126.7, 125.3, 122.9,
104.8, 96.5, 80.7, 78.9, 75.4, 68.9, 55.9, 55.5, 52.2, 51.8,
51.4, 44.7, 43.7, 40.6, 32.7, 7.9. ESIMS (m/z) 412.2 [M+1]+.
5.1.2. 3-Demethoxycarbonyl-3-hydroxylmethyl-3b,22-epox-
ide-4-deacetyl-vindoline (5). To a solution of triol 4 (3.86 g,
10 mmol) in 25 mL THF were added 50% NaOH solution
(4 g) and n-Bu4N+Iꢀ (36.9 mg, 0.1 mmol). After stirring at
50 ꢁC for 30 min, Toluene-4-sulfonyl chloride (2.09 g,
11 mmol) was added slowly. The mixture was stirred at
80 ꢁC for 3 h, and then was diluted with EtOAc
(50 mL)and H2O (30 mL). The organic layer was washed
with H2O (20 mL), brine (20 mL) and dried (Na2SO4). Re-
moval of the solvents was followed by column chromatog-
raphy on silica gel (7:1, hexanes/acetone) to afford the
5.1.4. 3-Demethoxycarbonyl-3-aminomethyl-4-deacetyl-
vindoline (7). To a suspension of LiAlH4 (800 mg,
21.06 mmol) in dry THF (20 mL) at 0 ꢁC, a solution
of compound 6 (2.89 g, 7.02 mmol) in dry THF
(20 mL) was added slowly. The reaction mixture was
stirred at room temperature for 4 h and then quenched
cautiously by the subsequent addition of water
(0.8 mL), 15% NaOH (aq) (0.8 mL), and water
compound 5 as a white solid in 80% (2.95 g) yield.
20
½aꢁ þ 4:0ꢂ (c 0.21, CHCl3), 1H NMR (CDCl3,
D

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Y. Shao et al. / Bioorg. Med. Chem. 15 (2007) 5061–5075
(2.4 mL). The resulting suspension was warmed to room
temperature and stirred for 10 min. The suspension was
filtered through a fritted funnel and the filtrate was con-
(CDCl3, 300 MHz): d: 9.05 (s), 6.86 (d, J = 8.1 Hz,
1H), 6.30 (dd, J = 8.1, 2.4 Hz, 1H), 6.14 (d, J = 2.4 Hz,
1H), 5.87 (dd, J = 10.2, 4.5 Hz, 1H), 5.34 (d,
J = 10.2 Hz, 1H), 5.26 (s, 1H), 5.00 (s, 1H), 4.09 (q,
J = 6.9 Hz, 1H), 3.78 (s, 3H), 3.51 (m, 1H), 3.43 (s,
1H), 3.35 (m, 2H), 3.11 (d, J = 12.3 Hz, 1H), 2.90 (s,
3H), 2.81 (d, J = 15.9 Hz, 1H), 2.62 (s, 1H), 2.50 (m,
1H), 2.26–2.17 (m, 2H), 2.10 (s, 3H), 1.27 (m, 1H),
1.22 (t, J = 6.9 Hz, 3H), 1.00 (m, 1H), 0.51 (t,
J = 7.5 Hz, 3H); 13C NMR (CDCl3, 75 MHz): d: 170.2,
160.8, 156.5, 154.1, 130.1, 125.5, 123.8, 122.4, 104.8,
96.4, 81.6, 76.9, 75.6, 67.2, 60.3, 55.0, 51.9, 51.3, 50.6,
44.8, 44.4, 42.6, 40.3, 31.1, 20.6, 14.4, 7.3. ESIMS
(m/z) 500.3 [M+1]+.
centrated in vacuo to give amine 7. The product was
20
D
used in the next step without purification. ½aꢁ þ 38ꢂ
1
(c 0.09, CHCl3), H NMR (CDCl3, 400 MHz) d: 8.64
(s, 1 H), 6.87 (d, J = 8.0 Hz, 1H), 6.28 (dd, J = 8.0,
2.4 Hz, 1H), 6.06 (d, J = 2.4 Hz, 1H), 5.87–5.79 (m,
2H), 3.78 (s, 4H), 3.45 (dd, J = 15.6, 4.4 Hz, 1H),
3.36–3.32 (m, 1H), 3.24 (s, 1H), 3.20 (d, J = 11.6 Hz,
1H), 3.16 (d, J = 11.6 Hz, 1H), 2.92 (s, 3H), 2.83 (dt,
J = 15.6, 2.0 Hz, 1H), 2.59 (s, 1H), 2.52–2.47 (m, 1H),
2.21–2.15 (m, 2H), 1.42–1.37 (m, 1H), 0.94–0.88 (m,
1H), 0.65 (t, J = 7.6 Hz, 3H); 13C NMR (CDCl3,
75 MHz) d: 160.8, 154.4, 131.9, 126.5, 122.9, 122.8,
104.2, 96.1, 84.5, 78.4, 75.8, 68.1, 55.4, 52.5, 51.5, 51.3,
49.7, 45.2, 43.6, 41.5, 32.4, 7.9. EIMS (m/z) 385 (M+),
368, 355, 297, 194, 174, 162, 152, 135, 122, 93.
5.2.3. 3-Demethoxycarbonyl-3-(isopropyloxycarbonyl-
amino)-methyl-vindoline (10a). Compound 10a was pre-
pared from isopropanol as material. Yield: 76%; 1H
NMR (CDCl3, 300 MHz): d: 9.01 (s), 6.78 (d,
J = 8.1 Hz, 1H), 6.21 (dd, J = 8.1, 1.8 Hz, 1H), 6.04 (d,
J = 1.8 Hz, 1H), 5.78 (dd, J = 10.2, 3.9 Hz, 1H), 5.27
(d, J = 10.2 Hz, 1H), 5.17 (d, J = 6.6 Hz, 1H), 4.92 (s,
1H), 4.79 (m, 1H), 3.67 (s, 3H), 3.40 (m, 2H), 3.35 (s,
1H), 3.24 (m, 1H), 3.02 (d, J = 12.6 Hz, 1H), 2.80 (s,
3H), 2.73 (d, J = 16.2 Hz, 1H), 2.54 (s, 1H), 2.42 (m,
1H), 2.22–2.11 (m, 2H), 2.00 (s, 3H), 1.20 (m, 1H),
1.11 (d, J = 6.0 Hz, 6H), 0.95 (m, 1H), 0.42 (t,
J = 7.2 Hz, 3H); 13C NMR (CDCl3, 75 MHz): d: 170.3,
160.8, 156.2, 154.2, 130.2, 125.5, 123.9, 122.4, 104.9,
96.4, 81.7, 77.0, 75.8, 67.5, 67.3, 55.1, 52.0, 51.4, 50.7,
44.9, 44.5, 42.7, 40.3, 31.2, 22.0 (2C), 20.7, 7.4. ESIMS
(m/z) 514.3 [M+1]+.
5.2. General procedure for the preparation of 8a–30a
A solution of the alcohol (1.50 mmol) in dry CH2Cl2
(10 ml) was treated with bis(trichloromethyl)carbonate
(147 mg, 0.50 mmol) followed by the dropwise addition
of
a solution of diisopropylethylamine (0.26 mL,
1.5 mmol) in CH2Cl2 (5 ml) at 0 ꢁC. This was allowed
to warm to room temperature and stirred a further 1 h
to obtain chloroformate solution in CH2Cl2. Then, the
prepared chloroformate was added dropwise to a solu-
tion of compound 7 (1 mmol) and diisopropylethyl-
amine (0.20 mL, 1.2 mmol) in dry CH2Cl2 (10 ml) at
room temperature. After stirring for 4 h, this mixture
was quenched by adding methanol (2 mL) with vigorous
stirring. This was extracted with CH2Cl2 (twice) and the
combined organic extracts were washed with brine and
dried (Na2SO4). The residue was added to pyridine
(1.0 mL) and Ac2O (1.0 mL) at room temperature. After
stirring the reaction mixture for 8 h, saturated NaHCO3
(5 mL) and EtOAc (20 mL) were added, and the organic
phase was washed with H2O (3· 10 mL) and brine
(10 mL), and was dried, concentrated, and purified by
flash chromatography (5:1 hexanes/acetone) to provide
a white solid (8a–30a).
5.2.4. 3-Demethoxycarbonyl-3-(tertbutyloxycarbonylami-
no)-methyl-vindoline (11a). Compound 11a was prepared
from di-tert-butyl dicarbonate as material. Yield: 86%;
1H NMR (CDCl3, 300 MHz): d: 9.01 (s), 6.79 (d,
J = 8.1 Hz, 1H), 6.21 (dd, J = 8.1, 1.8 Hz, 1H), 6.04 (d,
J = 1.8 Hz, 1H), 5.80 (dd, J = 10.2, 4.5 Hz, 1H), 5.28
(d, J = 10.2 Hz, 1H), 5.22 (s, 1H), 4.92 (s, 1H), 3.67 (s,
3H), 3.40 (m, 2H), 3.35 (s, 1H), 3.25 (m, 1H), 3.03 (d,
J = 12.3 Hz, 1H), 2.80 (s, 3H), 2.73 (d, J = 15.9 Hz,
1H), 2.54 (s, 1H), 2.43 (m, 1H), 2.22–2.06 (m, 2H),
2.00 (s, 3H), 1.36 (s, 9H), 1.20 (m, 1H), 0.90 (m, 1H),
0.50 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3, 75 MHz):
d: 170.3, 160.9, 156.4, 154.6, 130.8, 126.5, 124.3, 122.7,
104.6, 96.4, 81.6, 79.0, 76.6, 75.7, 68.5, 55.2, 51.9, 51.4,
50.6, 44.9, 44.5, 43.6, 40.5, 31.5, 28.4 (3C), 21.1, 7.7.
ESIMS (m/z) 528.4 [M+1]+.
5.2.1. 3-Demethoxycarbonyl-3-(methoxycarbonylamino)-
methyl-vindoline (8a). Compound 8a was prepared from
methyl chloroformate as material. Yield: 85%; 1H NMR
(CDCl3, 300 MHz): d: 9.04 (s), 6.84 (d, J = 8.1 Hz, 1H),
6.28 (dd, J = 8.1, 2.1 Hz, 1H), 6.12 (d, J = 2.1 Hz, 1H),
5.85 (dd, J = 10.2, 4.5 Hz, 1H), 5.34 (d, J = 10.2 Hz,
1H), 5.29 (s, 1H), 4.98 (s, 1H), 3.82 (s, 3H), 3.75 (s, 3H),
3.47 (m, 1H), 3.40 (s, 1H), 3.32 (m, 3H), 3.09 (d,
J = 12.6 Hz, 1H), 2.87 (s, 3H), 2.82 (d, J = 15.6 Hz, 1H),
2.59 (s, 1H), 2.49 (m, 1H), 2.30–2.12 (m, 2H), 2.08 (s,
3H), 1.27 (m, 1H), 0.98 (m, 1H), 0.49 (t, J = 7.5 Hz,
3H); 13C NMR (CDCl3, 75 MHz): d: 170.7, 161.1,
157.3, 154.5, 130.5, 125.8, 124.2, 122.7, 105.2, 96.8, 82.0,
77.2, 76.0, 67.5, 55.4, 52.3, 52.2, 51.7, 50.9, 45.2, 44.8,
42.9, 40.7, 31.5, 21.0, 7.6. ESIMS (m/z) 486.3 [M+1]+.
5.2.5. 3-Demethoxycarbonyl-3-(isobutyloxycarbonylami-
no)-methyl-vindoline (12a). Compound 12a was pre-
pared from iso-butyl chloroformate as material. Yield:
83%;1H NMR (CDCl3, 300 MHz): d: 9.01 (s), 6.79 (d,
J = 8.1 Hz, 1H), 6.21 (dd, J = 8.1, 1.8 Hz, 1H), 6.04 (d,
J = 1.8 Hz, 1H), 5.80 (dd, J = 10.2, 4.5 Hz, 1H), 5.28
(d, J = 10.2 Hz, 1H), 5.22 (s, 1H), 4.92 (s, 1H), 3.73 (d,
J = 6.3 Hz, 2H), 3.67 (s, 3H), 3.40 (m, 2H), 3.35 (s,
1H), 3.25 (m, 1H), 3.03 (d, J = 12.3 Hz, 1H), 2.80 (s,
3H), 2.73 (d, J = 15.9 Hz, 1H), 2.54 (s, 1H), 2.43 (m,
1H), 2.22–2.06 (m, 2H), 2.00 (s, 3H), 1.78 (m, 1H),
1.20 (m, 1H), 0.90 (m, 1H), 0.80 (d, J = 6.6 Hz, 6H),
0.42 (t, J = 6.9 Hz, 3H); 13C NMR (CDCl3, 75 MHz):
5.2.2. 3-Demethoxycarbonyl-3-(ethoxycarbonylamino)-
methyl-vindoline (9a). Compound 9a was prepared from
1
ethyl chloroformate as material. Yield: 81%; H NMR

Y. Shao et al. / Bioorg. Med. Chem. 15 (2007) 5061–5075
5067
d: 170.3, 160.8, 156.7, 154.2, 130.2, 125.5, 123.9, 122.4,
104.8, 96.4, 81.6, 77.0, 75.7, 70.6, 67.3, 55.1, 51.9, 51.4,
50.6, 44.9, 44.5, 42.6, 40.3, 31.2, 27.8, 20.7, 18.8 (2C),
7.3. ESIMS (m/z) 528.3 [M+1]+.
(d, J = 8.4 Hz, 1H), 6.30 (dd, J = 8.4, 2.4 Hz, 1H), 6.14
(d, J = 2.4 Hz, 1H), 5.87 (dd, J = 10.2, 4.5 Hz, 1H), 5.36
(d, J = 10.2 Hz, 2H), 5.00 (s, 1H), 3.86 (d, J = 7.2 Hz,
2H), 3.78 (s, 3H), 3.54–3.46 (m, 2H), 3.45 (s, 1H), 3.38–
3.31 (m, 1H), 3.12 (d, J = 12.6 Hz, 1H), 2.90 (s, 3H), 2.81
(d, J = 15.9 Hz, 1H), 2.62 (s, 1H), 2.56–2.46 (m, 1H),
2.28–2.18 (m, 2H), 2.11 (s, 3H), 1.34–1.26 (m, 1H), 1.13–
1.03 (m, 1H), 1.00–0.94 (m, 1H), 0.51 (t, J = 7.2 Hz, 3H),
0.51 (d, J = 4.8 Hz, 2H), 0.26 (d, J = 4.8 Hz, 2H); 13C
NMR (CDCl3, 75 MHz) d: 170.4, 160.9, 156.8, 154.4,
130.3, 125.6, 124.0, 122.6, 105.0, 96.5, 81.7, 77.1, 75.9,
69.4, 67.4, 55.2, 52.1, 51.5, 50.8, 45.0, 44.6, 42.8, 40.4,
31.3, 20.8, 10.1, 7.4, 3.0 (2· C). ESIMS (m/z) 526.3 [M+1]+.
5.2.6. 3-Demethoxycarbonyl-3-(2-methoxylethyloxycarbon-
ylamino)-methyl-vindoline (13a). Compound 13a was pre-
pared from 2-methoxylethanol as material. Yield: 65%;
1H NMR (CDCl3, 300 MHz): d: 8.98 (s), 6.81 (d,
J = 8.4 Hz, 1H), 6.24 (d, J = 8.4 Hz, 1H), 6.07 (s, 1H),
5.80 (dd, J = 10.2, 4.2 Hz, 1H), 5.39 (d, J = 7.2 Hz, 1H),
5.30 (d, J = 10.2 Hz, 1H), 4.94 (s, 1H), 4.14 (t,
J = 6.3 Hz, 2H), 3.71 (s, 3H), 3.50 (t, J = 6.3 Hz, 2H),
3.50–3.18 (m, 3H), 3.38 (s, 1H), 3.30 (s, 3H), 3.05 (d,
J = 12.6 Hz, 1H), 2.83 (s, 3H), 2.75 (d, J = 15.3 Hz, 1H),
2.56 (s, 1H), 2.45 (q, J = 9.0 Hz, 1H), 2.24–2.08 (m, 2H),
2.04 (s, 3H), 1.26–1.18 (m, 1H), 0.99–0.87 (m, 1H), 0.45
(t, J = 7.2 Hz, 3H); 13C NMR (CDCl3, 75 MHz): d:
170.5, 161.0, 156.5, 154.4, 130.3, 125.7, 124.0, 122.6,
105.1, 96.6, 81.7, 77.1, 75.9, 70.9, 67.5, 63.7, 58.8, 55.2,
52.1, 51.5, 50.8, 45.0, 44.6, 42.8, 40.5, 31.4, 20.8, 7.5.
ESIMS (m/z) 530.3 [M+1]+.
5.2.10. 3-Demethoxycarbonyl-3-(cyclobutyloxycarbonylami-
no)-methyl-vindoline (17a). Compound 17a was prepared
from cyclobutanol as material. Yield: 65%; 1H NMR
(CDCl3, 300 MHz): d: 9.06 (s), 6.86 (d, J = 8.4 Hz, 1H),
6.30 (dd, J = 8.4, 2.4 Hz, 1H), 6.14 (d, J = 2.4 Hz, 1H),
5.87 (dd, J = 10.2, 4.8 Hz, 1H), 5.36 (d, J = 10.2 Hz, 1H),
5.27 (d, J = 7.5 Hz, 1H), 4.99 (s, 1H), 4.90 (m, 1H), 3.78
(s, 3H), 3.48 (m, 2H), 3.43 (s, 1H), 3.35 (m, 1H), 3.09 (d,
J = 12.3 Hz, 1H), 2.89 (s, 3H), 2.80 (d, J = 15.9 Hz, 1H),
2.62 (s, 1H), 2.49 (m, 1H), 2.32–2.14 (m, 4H), 2.10 (s,
3H), 2.01 (m, 2H), 1.73 (m, 1H), 1.57 (m, 1H), 1.28 (m,
1H), 0.98 (m, 1H), 0.51 (t, J = 7.2 Hz, 3H); 13C NMR
(CDCl3, 75 MHz): d: 170.1, 160.7, 155.7, 154.1, 130.1,
125.4, 123.8, 122.4, 104.8, 96.4, 81.6, 76.8, 75.6, 68.3, 67.1,
54.9, 51.9, 51.2, 50.5, 44.7, 44.4, 42.5, 40.3, 31.1, 30.3,
30.1, 20.6, 12.9, 7.2. ESIMS (m/z) 526.4 [M+1]+.
5.2.7. 3-Demethoxycarbonyl-3-(bromoethyloxycarbonyl-
amino)-methyl-vindoline (14a). Compound 14a was pre-
pared from 2-Bromoethanol as material. Yield: 60%;
1H NMR (CDCl3, 300 MHz): d: 9.12 (s), 6.87 (d,
J = 8.4 Hz, 1H), 6.32 (dd, J = 8.4, 2.4 Hz, 1H), 6.15 (d,
J = 2.4 Hz, 1H), 5.89 (dd, J = 10.5, 3.6 Hz, 1H), 5.43
(m, 1H), 5.38 (d, J = 10.5 Hz, 1H), 5.01 (s, 1H), 4.35
(t, J = 6.3 Hz, 2H), 3.79 (s, 3H), 3.49 (t, J = 6.3 Hz,
2H), 3.52–3.30 (m, 3H), 3.43 (s, 1H), 3.14 (d,
J = 13.2 Hz, 1H), 2.91 (s, 3H), 2.83 (d, J = 14.7 Hz,
1H), 2.64 (s, 1H), 2.53 (q, J = 9.0 Hz, 1H), 2.25–2.10
(m, 2H), 2.12 (s, 3H), 1.26–1.18 (m, 1H), 1.10–0.87 (m,
1H), 0.52 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3,
75 MHz): d: 170.4, 161.0, 155.8, 154.3, 130.3, 125.6,
124.0, 122.6, 105.1, 96.6, 82.0, 77.0, 75.8, 67.4, 64.1,
55.2, 52.2, 51.5, 50.8, 45.2, 44.6, 42.8, 40.7, 31.3, 29.5,
20.9, 7.4. ESIMS (m/z) 578.2 [M+1]+.
5.2.11. 3-Demethoxycarbonyl-3-(cyclopentyloxycarbon-
ylamino)-methyl-vindoline (18a). Compound 18a was
prepared from cyclopentanol as material. Yield: 58%;
1H NMR (CDCl3, 300 MHz): d: 8.97 (s), 6.78 (d,
J = 8.4 Hz, 1H), 6.48 (dd, J = 8.4, 2.4 Hz, 1H), 6.04 (d,
J = 2.4 Hz, 1H), 5.79 (dd, J = 10.2, 4.8 Hz, 1H), 5.27
(d, J = 10.2 Hz, 1H), 5.14 (d, J = 7.5 Hz, 1H), 4.97 (m,
1H), 4.91 (s, 1H), 3.67 (s, 3H), 3.38 (m, 2H), 3.34 (s,
1H), 3.25 (m, 1H), 3.20 (d, J = 12.3 Hz, 1H), 2.80 (s,
3H), 2.73 (d, J = 15.6 Hz, 1H), 2.54 (s, 1H), 2.40 (m,
1H), 2.21–2.13 (m, 2H), 2.00 (s, 3H), 1.72 (m, 2H),
1.58 (m, 4H), 1.44 (m, 2H), 1.24 (m, 1H), 0.92 (m,
1H), 0.42 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3,
75 MHz): d: 170.2, 160.8, 156.4, 154.2, 130.2, 125.5,
123.9, 122.4, 104.8, 96.4, 81.7, 77.0 (2C), 75.7, 67.3,
55.0, 51.9, 51.4, 50.7, 44.9, 44.4, 42.6, 40.2, 32.6 (2C),
31.2, 23.4 (2C), 20.7, 7.3. ESIMS (m/z) 540.4 [M+1]+.
5.2.8. 3-Demethoxycarbonyl-3-(n-pentanyloxycarbonyl-
amino)-methyl-vindoline (15a). Compound 15a was pre-
pared from n-pentanol as material. Yield: 62%; 1H
NMR (CDCl3, 300 MHz): d: 9.05 (s), 6.82 (d,
J = 8.1 Hz, 1H), 6.25 (dd, J = 8.1, 2.1 Hz, 1H), 6.04 (d,
J = 2.1 Hz, 1H), 5.82 (dd, J = 10.2, 3.3 Hz, 1H), 5.31
(d, J = 10.2 Hz, 1H), 5.25 (d, J = 10.2 Hz, 1H), 4.96 (s,
1H), 3.97 (t, J = 6.3 Hz, 2H), 3.71 (s, 3H), 3.45 (m,
2H), 3.39 (s, 1H), 3.29 (m, 1H), 3.07 (d, J = 12.3 Hz,
1H), 2.84 (s, 3H), 2.77 (d, J = 15.9 Hz, 1H), 2.58 (s,
1H), 2.46 (m, 1H), 2.26–2.10 (m, 2H), 2.05 (s, 3H),
1.53 (m, 2H), 1.26(m, 5H), 0.93 (m, 1H), 0.74 (m, 5H),
0.46 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3, 75 MHz):
d: 170.4, 160.9, 156.9, 154.4, 130.3, 125.6, 124.0, 122.6,
105.0, 96.5, 81.8, 77.1, 75.9, 67.4, 64.8, 55.2, 52.1, 51.6,
50.8, 45.0, 44.6, 42.8, 40.5, 31.3, 28.7, 27.9, 22.3, 20.9,
14.0, 7.5. ESIMS (m/z) 542.4 [M+1]+.
5.2.12. 3-Demethoxycarbonyl-3-(cyclohexanyloxycarbon-
ylamino)-methyl-vindoline (19a). Compound 19a was
prepared from cyclohexanol as material. Yield: 56%;
1H NMR (CDCl3, 300 MHz): d: 8.97 (s), 6.79 (d,
J = 8.4 Hz, 1H), 6.22 (dd, J = 8.4, 2.4 Hz, 1H), 6.06 (d,
J = 2.4 Hz, 1H), 5.79 (dd, J = 10.2, 4.8 Hz, 1H), 5.29
(d, J = 10.2 Hz, 1H), 5.19 (d, J = 5.7 Hz, 1H), 4.93 (s,
1H), 4.53 (m, 1H), 3.69 (s, 3H), 3.40 (m, 2H), 3.37 (s,
1H), 3.27 (m, 1H), 3.04 (d, J = 12.3 Hz, 1H), 2.82 (s,
3H), 2.74 (d, J = 15.6 Hz, 1H), 2.55 (s, 1H), 2.42 (m,
1H), 2.25–2.15 (m, 2H), 2.02 (s, 3H), 1.75 (m, 2H),
1.62 (m, 2H), 1.42 (m, 1H), 1.26 (m, 6H), 0.88 (m,
1H), 0.44 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3,
75 MHz): d: 170.3, 160.9, 156.3, 154.3, 130.3, 125.6,
5.2.9. 3-Demethoxycarbonyl-3-(cyclopropylmethyloxy-
carbonylamino)-methyl-vindoline (16a). Compound 16a
was prepared from cyclopropylmethanol as material.
Yield: 63%; 1H NMR (CDCl3, 300 MHz) d: 9.07 (s), 6.86

5068
Y. Shao et al. / Bioorg. Med. Chem. 15 (2007) 5061–5075
124.0, 122.5, 104.9, 96.4, 81.7, 77.1, 75.8, 72.6, 67.4,
55.1, 52.0, 51.5, 50.7, 45.0, 44.5, 42.7, 40.3, 31.9, 31.3
(2C), 25.5, 23.7 (2C), 20.8, 7.4. ESIMS (m/z) 554.4
[M+1]+.
NMR (CDCl3, 300 MHz): d: 9.10 (s), 7.30 (m, 5H),
6.86 (d, J = 8.4 Hz, 1H), 6.30 (dd, J = 8.4, 2.4 Hz, 1H),
6.13 (d, J = 2.4 Hz, 1H), 5.87 (dd, J = 10.2, 4.5 Hz,
1H), 5.43 (d, J = 7.5 Hz, 1H), 5.36 (d, J = 10.2 Hz,
1H), 5.09 (s, 2H), 5.01 (s, 1H), 3.77 (s, 3H), 3.50 (m,
2H), 3.42 (s, 1H), 3.34 (m, 1H), 3.14 (d, J = 12.6 Hz,
1H), 2.88 (s, 3H), 2.81 (d, J = 15.9 Hz, 1H), 2.62 (s,
1H), 2.49 (m, 1H), 2.26–2.16 (m, 2H), 2.08 (s, 3H),
1.28 (m, 1H), 1.00 (m, 1H), 0.51 (t, J = 7.5 Hz, 3H);
13C NMR (CDCl3, 75 MHz): d: 170.6, 161.1, 156.6,
154.5, 136.8, 130.4, 128.5 (2C), 128.1 (2C), 125.7,
124.2, 122.7, 105.2, 96.7, 81.9, 77.2, 76.0, 67.5, 66.6,
55.4, 52.2, 51.6, 50.9, 45.2, 44.7, 42.9, 40.6, 31.4, 21.0,
7.6. ESIMS (m/z) 562.3 [M+1]+.
5.2.13. 3-Demethoxycarbonyl-3-(phenyloxycarbonylami-
no)-methyl-vindoline (20a). Compound 20a was prepared
from phenol as material. Yield: 65%; 1H NMR (CDCl3,
300 MHz): d: 9.18 (s), 7.30 (t, J = 7.2 Hz, 2H), 7.01 (m,
3H), 6.86 (dd, J = 8.4, 1.8 Hz, 1H), 6.30 (dd, J = 8.4,
1.8 Hz, 1H), 6.14 (s, 1H), 5.87 (dd, J = 10.2, 4.5 Hz,
1H), 5.76 (d, J = 7.5 Hz, 1H), 5.37 (d, J = 9.9 Hz, 1H),
5.01 (s, 1H), 3.74 (s, 3H), 3.60 (m, 1H), 3.47 (s, 1H),
3.42(m, 1H), 3.34 (m, 1H), 3.22 (d, J = 12.6 Hz, 1H),
2.88 (s, 3H), 2.80 (d, J = 15.9 Hz, 1H), 2.65 (s, 1H),
2.49 (m, 1H), 2.26–2.16 (m, 2H), 2.10 (s, 3H), 1.28 (m,
1H), 1.00 (m, 1H), 0.52 (t, J = 7.5 Hz, 3H); 13C NMR
(CDCl3, 75 MHz): d: 170.5, 160.9, 154.6, 154.2, 151.0,
130.2, 129.1 (2C), 125.5, 125.0, 124.0, 122.6, 121.5
(2C), 105.3, 96.7, 82.3, 76.8, 75.8, 67.2, 55.2, 52.2,
51.3, 50.7, 45.2, 44.5, 42.7, 40.9, 31.2, 20.8, 7.4. ESIMS
(m/z) 548.3 [M+1]+.
5.2.17. 3-Demethoxycarbonyl-3-(2-methoxylbenzyloxy-
carbonylamino)-methyl-vindoline (24a). Compound 24a
was prepared from 2-methoxyl benzyl alcohol as mate-
1
rial. Yield: 78%; H NMR (CDCl3, 300 MHz): d: 9.10
(s), 7.23 (d, J = 7.2 Hz, 1H), 7.16 (t, J = 7.2 Hz, 1H),
6.82 (t, J = 7.2 Hz, 1H), 6.77 (d, J = 8.4 Hz, 1H), 6.76
(d, J = 7.2 Hz, 1H), 6.21 (dd, J = 8.4, 2.4 Hz, 1H), 6.04
(s, 1H), 5.77 (dd, J = 10.2, 3.9 Hz, 1H), 5.35 (d,
J = 6.3 Hz, 1H), 5.27 (d, J = 10.2 Hz, 1H), 5.07 (s,
2H), 4.93 (s, 1H), 3.70 (s, 3H), 3.66 (s, 3H), 3.47–3.32
(m, 2H), 3.35 (s, 1H), 3.27–3.19 (m, 1H), 3.07 (d,
J = 12.9 Hz, 1H), 2.79 (s, 3H), 2.71 (d, J = 15.9 Hz,
1H), 2.52 (s, 1H), 2.42–2.36 (m, 1H), 2.20–2.08 (m,
2H), 1.91 (s, 3H), 1.33–1.22 (m, 1H), 0.95–0.86 (m,
1H), 0.42 (t, J = 7.5 Hz, 3H); 13C NMR (CDCl3,
75 MHz): d: 170.4, 160.9, 157.2, 156.6, 154.4, 130.3,
129.3, 129.2, 125.6, 124.9, 124.0, 122.5, 120.3, 110.2,
105.0, 96.5, 81.7, 77.1, 75.9, 67.5, 61.8, 55.2 (2C), 52.0,
51.5, 50.7, 45.2, 44.5, 42.8, 40.4, 31.3, 20.7, 7.4. ESIMS
(m/z) 592.2 [M+1]+.
5.2.14. 3-Demethoxycarbonyl-3-(2-methoxyl-phenyloxy-
carbonylamino)-methyl-vindoline (21a). Compound 21a
was prepared from 2-methoxyl-phenol as material.
1
Yield: 60%; H NMR (CDCl3, 300 MHz): d: 9.16 (s),
7.15 (t, J = 7.2 Hz, 1H), 7.07(d, J = 7.2 Hz, 1H), 7.01
(m, 3H), 6.94–6.87 (m, 3H), 6.31 (dd, J = 8.4, 1.8 Hz,
1H), 6.15 (s, 1H), 5.89 (dd, J = 10.2, 4.5 Hz, 1H), 5.78
(d, J = 8.1 Hz, 1H), 5.38 (d, J = 10.5 Hz, 1H), 5.03 (s,
1H), 3.78 (s, 6H), 3.60 (m, 1H), 3.53 (s, 1H), 3.47(m,
1H), 3.35 (m, 1H), 3.22 (d, J = 12.6 Hz, 1H), 2.88 (s,
3H), 2.82 (d, J = 15.9 Hz, 1H), 2.66 (s, 1H), 2.52 (m,
1H), 2.26–2.16 (m, 2H), 2.12 (s, 3H), 1.28 (m, 1H),
1.00 (m, 1H), 0.53 (t, J = 7.5 Hz, 3H); 13C NMR
(CDCl3, 75 MHz): d: 170.6, 161.9, 154.5 (2C), 151.8,
140.1, 130.4, 126.5, 125.7, 124.2, 123.4, 122.7, 120.7,
112.3, 105.3, 96.7, 82.0, 77.0, 76.1, 67.4, 55.8, 55.4,
52.3, 51.6, 50.9, 45.3, 44.7, 42.9, 40.7, 31.4, 21.0, 7.6.
ESIMS (m/z) 578.2 [M+1]+.
5.2.18. 3-Demethoxycarbonyl-3-(4-methoxylbenzyloxy-
carbonylamino)-methyl-vindoline (25a). Compound 25a
was prepared from 4-methoxyl benzyl alcohol as mate-
1
rial. Yield: 76%; H NMR (CDCl3, 300 MHz): d: 8.95
(s), 7.19 (d, J = 8.4 Hz, 2H), 6.78 (d, J = 8.4 Hz, 2H),
6.76 (d, J = 8.1 Hz, 1H), 6.21 (dd, J = 8.1, 1.8 Hz, 1H),
6.04 (s, 1H), 5.77 (dd, J = 10.2, 4.5 Hz, 1H), 5.32 (d,
J = 6.3 Hz, 1H), 5.27 (d, J = 10.2 Hz, 1H), 4.93 (s,
3H), 3.67 (s, 6H), 3.43 - 3.38 (m, 2H), 3.33 (s, 1H),
3.26–3.20 (m, 1H), 3.06 (d, J = 12.3 Hz, 1H), 2.78 (s,
3H), 2.71 (d, J = 16.2 Hz, 1H), 2.53 (s, 1H), 2.40 (q,
J = 8.7 Hz, 1H), 2.16–2.06 (m, 2H), 1.98 (s, 3H), 1.25–
1.17 (m, 1H), 0.94–0.87 (m, 1H), 0.43 (t, J = 7.2 Hz,
3H); 13C NMR (CDCl3, 75 MHz): d: 170.4, 160.9,
159.4, 156.4, 154.3, 130.3, 129.8 (2C), 128.7, 125.6,
124.0, 122.5, 113.7 (2C), 105.0, 96.5, 81.7, 77.0, 75.8,
67.5, 66.2, 55.2, 55.1, 52.1, 51.5, 50.7, 45.1, 44.5, 42.8,
40.4, 31.3, 20.8, 7.4. ESIMS (m/z) 592.2 [M+1]+.
5.2.15. 3-Demethoxycarbonyl-3-(4-methoxyl-phenyloxy-
carbonylamino)-methyl-vindoline (22a). Compound 22a
was prepared from 4-methoxyl-phenol as material.
1
Yield: 61%; H NMR (CDCl3, 300 MHz): d: 9.19 (s),
7.04 (d, J = 7.2 Hz, 2H), 6.87 (m, 3H), 6.33 (dd,
J = 8.4, 1.8 Hz, 1H), 6.16 (s, 1H), 5.89 (dd, J = 10.2,
4.5 Hz, 1H), 5.70 (d, J = 7.8 Hz, 1H), 5.38 (d,
J = 10.2 Hz, 1H), 5.03 (s, 1H), 3.78 (s, 6H), 3.61 (m,
1H), 3.48 (m, 1H), 3.47(s, 1H), 3.37 (m, 1H), 3.22 (d,
J = 12.6 Hz, 1H), 2.95 (s, 3H), 2.84 (d, J = 15.9 Hz,
1H), 2.67 (s, 1H), 2.52 (m, 1H), 2.26–2.16 (m, 2H),
2.12 (s, 3H), 1.28 (m, 1H), 1.00 (m, 1H), 0.53 (t,
J = 7.5 Hz, 3H); 13C NMR (CDCl3, 75 MHz): d: 170.6,
161.0, 156.8, 155.1, 154.3, 144.6, 130.3, 125.6, 124.2,
122.7, 122.4(2C), 114.2 (2C), 105.3, 96.8, 82.3, 76.8,
75.8, 67.2, 55.5, 55.3, 52.3, 51.4, 50.8, 45.3, 44.7, 42.8,
41.0, 31.3, 21.0, 7.5. ESIMS (m/z) 578.2 [M+1]+.
5.2.19. 3-Demethoxycarbonyl-3-(piperonyloxycarbonyl-
amino)-methyl-vindoline (26a). Compound 26a was pre-
pared from piperonyl alcohol as material. Yield: 69%;
1H NMR (CDCl3, 300 MHz): d: 9.00 (s), 6.83–6.67 (m.
4H), 6.25 (dd, J = 8.1, 1.8 Hz, 1H), 6.08 (s, 1H), 5.84
(s, 2H), 5.80 (dd, J = 10.2, 4.5 Hz, 1H), 5.39 (d,
J = 6.0 Hz, 1H), 5.31 (d, J = 10.2 Hz, 1H), 4.96 (s,
1H), 4.92 (s, 2H), 3.70 (s, 6H), 3.50–3.42 (m, 2H), 3.37
5.2.16. 3-Demethoxycarbonyl-3-(benzyloxycarbonylami-
no)-methyl-vindoline (23a). Compound 23a was prepared
1
from benzyl chloroformate as material. Yield: 85%; H

Y. Shao et al. / Bioorg. Med. Chem. 15 (2007) 5061–5075
5069
(s, 1H), 3.31–3.25 (m, 1H), 3.10 (d, J = 12.3 Hz, 1H),
2.82 (s, 3H), 2.76 (d, J = 16.2 Hz, 1H), 2.57 (s, 1H),
2.45 (q, J = 9.6 Hz, 1H), 2.24–2.09 (m, 2H), 2.02 (s,
3H), 1.28–1.20 (m, 1H), 0.98–0.89 (m, 1H), 0.46 (t,
J = 7.2 Hz, 3H); 13C NMR (CDCl3, 75 MHz): d: 170.3,
160.9, 156.3, 154.3, 147.6, 147.3, 130.4, 130.2, 125.6,
123.9, 122.5, 121.8, 108.7, 108.0, 104.9, 100.9, 96.5,
81.7, 77.0, 75.8, 67.4, 66.3, 55.1, 52.0, 51.4, 50.7, 45.1,
44.4, 42.7, 40.4, 31.3, 20.7, 7.4. ESIMS (m/z) 606.2
[M+1]+.
133.4, 130.3, 128.6, 128.4, 125.6, 124.9, 124.1, 122.7,
105.2, 96.7, 82.1, 77.0, 75.9, 67.4, 63.1, 55.3, 52.2, 51.5,
50.8, 45.2, 44.6, 42.8, 40.7, 31.4, 20.9, 7.5. ESIMS
(m/z) 607.3 [M+1]+.
5.2.23. 3-Demethoxycarbonyl-3-(4-nitro-benzyloxycarbon-
ylamino)-methyl-vindoline (30a). Compound 30a was
prepared from 4-nitrobenzyl alcohol as material. Yield:
1
61%; H NMR (CDCl3, 300 MHz): d: 9.20 (s), 8.21 (d,
J = 8.7 Hz, 2H), 8.50 (d, J = 8.7 Hz, 2H), 6.88 (d,
J = 8.4 Hz, 1H), 6.32 (dd, J = 8.4, 2.4 Hz, 1H), 6.15 (s,
1H), 5.90 (dd, J = 10.2, 3.6 Hz, 1H), 5.52 (d,
J = 7.5 Hz, 1H), 5.38 (d, J = 10.2 Hz, 1H), 5.19 (s,
2H), 5.01 (s, 1H), 3.79 (s, 3H), 3.58–3.33 (m, 3H), 3.42
(s, 1H), 3.16 (d, J = 12.9 Hz, 1H), 2.89 (s, 3H), 2.87 (d,
J = 16.2 Hz, 1H), 2.65 (s, 1H), 2.58–2.49 (m, 1H),
2.33–2.23 (m, 2H), 2.09 (s, 3H), 1.34–1.25 (m, 1H),
1.03–0.96 (m, 1H), 0.53 (t, J = 7.2 Hz, 3H); 13C NMR
(CDCl3, 75 MHz): d: 170.4, 160.9, 155.8, 154.1, 147.3,
144.2, 130.2, 127.9 (2C), 125.5, 124.0, 123.5 (2C),
122.6, 105.1, 96.6, 82.0, 76.9, 75.7, 67.3, 64.9, 55.1,
52.1, 51.3, 50.7, 45.2, 44.5, 42.7, 40.6, 31.2, 20.7, 7.4.
ESIMS (m/z) 607.2 [M+1]+.
5.2.20. 3-Demethoxycarbonyl-3-(3-chlorobenzyloxycarbon-
ylamino)-methyl-vindoline (27a). Compound 27a was
prepared from 3-chlorobenzyl alcohol as material.
1
Yield: 78%; H NMR (CDCl3, 300 MHz): d: 9.11 (s),
7.34 (s, 1H), 7.27–7.20 (m. 2H), 6.86 (d, J = 8.4 Hz,
1H), 6.31 (d, J = 8.4 Hz, 1H), 6.14 (s, 1H), 5.88 (dd,
J = 10.2, 4.5 Hz, 1H), 5.44 (d, J = 6.0 Hz, 1H), 5.37 (d,
J = 10.2 Hz, 1H), 5.06 (s, 2H), 5.01 (s, 1H), 3.79 (s,
3H), 3.56–3.34 (m, 3H), 3.42 (s, 1H), 3.15 (d,
J = 12.9 Hz, 1H), 2.88 (s, 3H), 2.82 (d, J = 16.2 Hz,
1H), 2.64 (s, 1H), 2.56–2.47 (m, 1H), 2.33–2.17 (m,
2H), 2.09 (s, 3H), 1.37–1.28 (m, 1H), 1.06–0.94 (m,
1H), 0.52 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3,
75 MHz): d: 170.4, 161.0, 156.1, 154.3, 138.8, 134.2,
130.3, 129.7, 128.0, 127.7, 125.7, 125.5, 124.0, 122.6,
105.1, 96.6, 81.9, 77.0, 75.9, 67.4, 65.4, 55.2, 52.1, 51.4,
50.8, 45.2, 44.6, 42.8, 40.5, 31.3, 20.8, 7.4. ESIMS
(m/z) 596.2 [M+1]+.
5.2.24. 3-Demethoxycarbonyl-3-(20-oxo-3,50-spirooxazoli-
dino)-methyl-vindoline (31a). To a solution of compound
20a (1 mmol, 574 mg) in dry THF (10 mL) was added
sodium hydride (60% in oil, 48 mg, 1.2 mmol) at room
temperature under a nitrogen atmosphere. After stirred
for 2 h, the reaction mixture was washed with saturated
ammonium chloride solution and then extracted with
EtOAc (twice) and the combined organic extracts were
washed with brine, dried (Na2SO4), concentrated, and
purified by flash chromatography (2:1 hexanes/acetone)
to provide a white solid (180 mg, 40%). 1H NMR
(CDCl3, 300 MHz): d: 6.83 (d, J = 7.8 Hz, 1H), 6.12
(d, J = 7.8 Hz, 1H), 5.96 (s, 1H), 5.87 (dd, J = 9.9,
4.8 Hz, 1H), 5.30 (d, J = 9.9 Hz, 1H), 5.17 (s, 1H),
4.90 (s, 1H), 3.81 (s, 1H), 3.78 (s, 3H), 3.40 (dd,
J = 16.5, 4.8 Hz, 1H), 3.18 (s, 2H), 3.21–3.12 (m, 2H),
3.10 (s, 3H), 2.69 (d, J = 16.5 Hz, 1H), 2.65 (s, 1H),
2.39–2.29 (m, 1H), 2.19–2.10 (m, 1H), 2.04 (s, 3H),
1.62–1.52 (m, 1H), 1.30–1.18 (m, 1H), 0.88 (t,
J = 7.2 Hz, 3H); 13C NMR (CDCl3, 75 MHz): d: 170.3,
160.9, 158.4, 151.1, 128.2, 127.9, 126.4, 120.4, 100.9,
92.5, 87.1, 75.2, 74.8, 62.5, 55.0, 52.9, 52.3, 51.6, 45.2,
44.4, 43.4, 33.5, 28.5, 20.9, 8.5. ESIMS (m/z) 454.1
[M+1]+.
5.2.21. 3-Demethoxycarbonyl-3-(4-chlorobenzyloxycarbon-
ylamino)-methyl-vindoline (28a). Compound 28a was
prepared from 4-Chlorobenzyl alcohol as material.
1
Yield: 78%; H NMR (CDCl3, 300 MHz): d: 9.01 (s),
7.29 (m. 4H), 6.87 (d, J = 8.4 Hz, 1H), 6.31 (d,
J = 8.4 Hz, 1H), 6.14 (s, 1H), 5.87 (dd, J = 10.2,
4.5 Hz, 1H), 5.42 (d, J = 6.6 Hz, 1H), 5.36 (d,
J = 10.2 Hz, 1H), 5.05 (s, 2H), 5.00 (s, 1H), 3.79 (s,
3H), 3.60–3.25 (m, 3H), 3.41 (s, 1H), 3.14 (d,
J = 12.6 Hz, 1H), 2.88 (s, 3H), 2.84 (d, J = 16.2 Hz,
1H), 2.63 (s, 1H), 2.56–2.42 (m, 1H), 2.31–2.14 (m,
2H), 2.09 (s, 3H), 1.40–1.20 (m, 1H), 1.04–0.96 (m,
1H), 0.52 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3,
75 MHz): d: 170.2, 160.9, 156.1, 154.2, 135.2, 133.5,
130.2, 129.2 (2C), 128.4 (2C), 125.5, 123.9, 122.5,
105.0, 96.5, 81.8, 76.9, 75.7, 67.3, 65.4, 55.0, 52.0, 51.4,
50.6, 45.1, 44.4, 42.7, 40.4, 31.2, 20.6, 7.3. ESIMS
(m/z) 596.2 [M+1]+.
5.2.22. 3-Demethoxycarbonyl-3-(2-nitro-benzyloxycarbon-
ylamino)-methyl-vindoline (29a). Compound 29a was
prepared from 2-nitrobenzyl alcohol as material. Yield:
5.2.25. 3-Demethoxycarbonyl-3-(isobutylcarbamoyloxy)-
methyl-vindoline (32a). To a cooled (cold water bath)
solution of CDI (2.2 mmol) in CH2Cl2 (5 mL) was
added the isobutyl amine (2.0 mmol) dropwise. After
the solids dissolved, the water bath was removed, and
the mixture stirred for a further 24 h. The reaction mix-
ture was diluted with CH2Cl2 (5 mL) and quenched with
water (20 mL). The aqueous layer was extracted with
CH2Cl2 (4· 5 mL), the combined organic layers dried
over anhydrous MgSO4, filtered, and concentrated in
vacuo to yield carbamoylimidazoles 30, which were used
in the next reaction without purification. Then, to a
solution of compound 4 (1 mmol, 386 mg) and 30 in
dry THF (10 mL) was added sodium hydride (60% in
1
64%; H NMR (CDCl3, 300 MHz): d: 9.11 (s), 7.97 (d,
J = 8.1 Hz, 1H), 7.52 (m, 2H), 7.36 (m. 1H), 6.80 (d,
J = 8.4 Hz, 1H), 6.23 (d, J = 8.4 Hz, 1H), 6.06 (s, 1H),
5.81 (dd, J = 10.2, 4.5 Hz, 1H), 5.50 (d, J = 7.5 Hz,
1H), 5.42 (s, 2H), 5.30 (d, J = 10.2 Hz, 1H), 4.94 (s,
1H), 3.68 (s, 3H), 3.49–3.25 (m, 3H), 3.37 (s, 1H), 3.08
(d, J = 12.9 Hz, 1H), 2.81 (s, 3H), 2.76 (d, J = 16.2 Hz,
1H), 2.58 (s, 1H), 2.50–2.41 (m, 1H), 2.24–2.12 (m,
2H), 2.01 (s, 3H), 1.25–1.17 (m, 1H), 0.98–0.84 (m,
1H), 0.44 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3,
75 MHz): d: 170.5, 161.0, 155.9, 154.3, 147.3, 133.7,

5070
Y. Shao et al. / Bioorg. Med. Chem. 15 (2007) 5061–5075
oil, 80 mg, 2 mmol) at room temperature under a nitro-
gen atmosphere. After stirring for 2 h, the reaction mix-
ture was washed with saturated ammonium chloride
solution and then extracted with CH2Cl2 (twice) and
the combined organic extracts were washed with brine
and dried (Na2SO4). The residue was added to pyridine
(1.0 mL) and Ac2O (1.0 mL) at room temperature. After
stirring the reaction mixture for 8h, saturated NaHCO3
(5 mL) and EtOAc (20 mL) were added, and the organic
phase was washed with H2O (3· 10 mL) and brine
(10 mL), and was dried, concentrated, and purified by
flash chromatography (4:1 hexanes/acetone) to provide
a white solid (275 mg, 52%). 1H NMR (CDCl3,
300 MHz): d: 9.10 (s), 6.86 (d, J = 8.4 Hz, 1H), 6.30
(d, J = 8.4 Hz, 1H), 6.12 (s, 1H), 5.89 (dd, J = 10.2,
2.4 Hz, 1H), 5.36 (d, J = 10.2 Hz, 1H), 5.04 (s, 1H),
4.89 (m, 1H), 4.10(s, 2H), 3.79 (s, 3H), 3.63 (s, 1H),
3.52–3.37 (m, 2H), 3.18 (q, J = 6.9 Hz, 1H), 2.88 (s,
3H), 2.82 (d, J = 15.9 Hz, 1H), 2.63 (s, 1H), 2.50 (q,
J = 9.3 Hz, 1H), 2.31–2.26 (m, 2H), 2.14 (s, 3H), 1.52–
1.41 (m, 2H), 1.38–1.22 (m, 3H), 1.05–0.98 (m, 1H),
1.02 (m, 1H), 0.91 (t, J = 7.2 Hz, 3H), 0.52 (t,
J = 7.5 Hz, 3H); 13C NMR (CDCl3, 75 MHz): d: 170.4,
160.7, 156.0, 154.1, 129.9, 125.6, 123.9, 122.4, 104.4,
95.9, 81.1, 76.4, 75.9, 67.5, 66.7, 54.9, 51.6, 51.5, 50.5,
44.3, 42.4, 39.4, 31.6, 31.1, 20.6, 19.5, 13.4, 7.3. ESIMS
(m/z) 528.2 [M+1]+.
34.5, 33.0, 31.6, 28.0, 25.7, 21.1, 12.4, 8.4; ESIMS
(m/e) 822.4 [M+1]+. HRESIMS C47H60N5O8 [M+H]+
calcd for 822.4442, found 822.4439.
5.3.2. 3-Demethoxycarbonyl-3-(ethoxycarbonylamino)-
20
D
methyl-anhydrovinblastine (9b). ½aꢁ þ 61:2ꢂ (c 0.56,
1
CHCl3); H NMR (CDCl3, 300 MHz): d: 9.25 (s, 1H),
8.00 (s, 1H), 7.53 (d, J = 7.5 Hz, 1H), 7.13 (m, 3H),
6.61 (s, 1H), 6.20 (s, 1H), 5.87 (dd, J = 10.2, 3.9 Hz,
1H), 5.48 (d, J = 6.3 Hz, 1H), 5.42 (d, J = 10.2 Hz,
1H), 5.30 (d, J = 3.9 Hz, 1H), 5.03 (s, 1H), 4.09 (q,
J = 7.2 Hz, 2H), 3.83 (s, 3H), 3.62 (s, 3H), 3.40 (s,
1H), 2.94 (s, 3H), 2.82 (d, J = 15.9 Hz, 1H), 2.61 (s,
1H), 2.16 (s, 3H), 1.43 (m, 1H), 1.23 (t, J = 7.2 Hz,
3H), 1.21 (m, 1H), 1.00 (t, J = 7.5 Hz, 3H), 0.81 (t,
J = 6.9 Hz, 3H); 13C NMR (CDCl3, 75 MHz): d: 174.4,
170.8, 157.8, 156.9, 153.8, 137.7, 134.9, 130.8, 129.8,
129.0, 124.7, 123.9, 123.8, 123.3, 122.8, 120.7, 119.4,
118.2, 115.6, 110.6, 95.0, 81.8, 77.0, 75.9, 65.9, 60.7,
55.9, 55.3, 54.4, 52.5, 52.5, 50.9, 50.1, 50.0, 45.4, 45.2,
44.6, 42.7, 40.4, 34.2, 31.5, 31.5, 27.8, 23.2, 21.0, 14.7,
12.4, 8.4; ESIMS (m/e) 836.4 [M+1]+. HRESIMS
C48H62N5O8 [M+H]+ calcd for 836.4598, found
836.4594.
5.3.3. 3-Demethoxycarbonyl-3-(isopropyloxycarbonyl-
20
D
amino)-methyl-anhydrovinblastine (10b). ½aꢁ þ 64:2ꢂ
1
(c 0.89, CHCl3); H NMR (CDCl3, 300 MHz): d: 9.24
(s, 1H), 8.00 (s, 1H), 7.53 (d, J = 7.5 Hz, 1H), 7.18 (m,
3H), 6.60 (s, 1H), 6.20 (s, 1H), 5.87 (dd, J = 10.2,
3.9 Hz, 1H), 5.47 (d, J = 6.0 Hz, 1H), 5.42 (d,
J = 10.2 Hz, 1H), 5.25 (d, J = 3.9 Hz, 1H), 5.03 (s,
1H), 4.89 (m, 1H), 3.83 (s, 3H), 3.64 (s, 3H), 3.38 (s,
1H), 2.93 (s, 3H), 2.82 (d, J = 15.9 Hz, 1H), 2.61 (s,
1H), 2.16 (s, 3H), 1.93 (q, J = 7.5 Hz, 2H), 1.43 (m,
1H), 1.25 (m, 1H), 1.23 (d, J = 7.2 Hz, 6H), 1.00 (t,
J = 7.5 Hz, 3H), 0.81 (t, J = 6.9 Hz, 3H); 13C NMR
(CDCl3, 75 MHz): d: 174.9, 171.0, 158.1, 156.7, 153.8,
139.9, 135.1, 131.0, 130.1, 129.6, 124.7, 124.0, 123.9,
123.7, 122.5, 121.7, 119.0, 118.5, 117.3, 110.6, 95.2,
82.2, 77.3, 76.1, 68.0, 66.2, 56.0, 55.6, 54.6, 52.7, 52.5,
52.2, 50.3 (2C), 46.0, 45.3, 44.9, 42.9, 40.7, 34.5, 32.9,
31.7, 28.0, 25.7, 22.3 (2C), 21.1, 12.4, 8.4; ESIMS
(m/e) 850.4 [M+1]+. HRESIMS C49H64N5O8 [M+H]+
calcd for 850.4755, found 850.4758.
5.3. General procedure for the preparation of 8b–32b
Catharanthine Tartrate (486 mg, 1 mmol) and ferric
chloride anhydrous (486 mg, 3 mmol) were combined
in a mixture of glycine buffer (containing 320 mg of gly-
cine and 250 mg of sodium chloride in 40 mL of water)
and hydrochloric acid (40 mL, 0.1 N) under a nitrogen
atmosphere. After 10 min of stirring at room tempera-
ture, compound 8a–32a (1 mmol) was added. After 8 h
of stirring at room temperature, sodium borohydride
(80 mg) in ammonium hydroxide (8 mL) was added
dropwise at 0 ꢁC and it was left to react for 15 min.
The reaction mixture was extracted with CH2Cl2 (4·
20 mL), and the organic phase was filtered with Celite
and concentrated at reduced pressure, then the residue
was followed by column chromatography on silica gel
(100:1, CHCl3/MeOH) to afford the compounds 8b–
32b as white solid in over 50% yield.
5.3.4. 3-Demethoxycarbonyl-3-(tertbutyloxycarbonyl-
20
D
5.3.1. 3-Demethoxycarbonyl-3-(methoxycarbonylamino)-
amino)-methyl-anhydrovinblastine (11b). ½aꢁ þ 63:1ꢂ
20
D
1
methyl-anhydrovinblastine (8b). ½aꢁ þ 60:3ꢂ (c 0.74,
(c 0.58, CHCl3); H NMR (CDCl3, 300 MHz): d: 9.18
(s, 1H), 8.01 (s, 1H), 7.51 (d, J = 7.2 Hz, 1H), 7.12 (m,
3H), 6.60 (s, 1H), 6.20 (s, 1H), 5.87 (dd, J = 10.2,
3.9 Hz, 1H), 5.47 (d, J = 6.0 Hz, 1H), 5.42 (d,
J = 10.2 Hz, 1H), 5.17 (d, J = 3.9 Hz, 1H), 5.03 (s,
1H), 3.82 (s, 3H), 3.60 (s, 3H), 3.40 (s, 1H), 2.95 (s,
3H), 2.79 (d, J = 15.9 Hz, 1H), 2.60 (s, 1H), 2.15 (s,
3H), 1.93 (q, J = 7.5 Hz, 2H), 1.43 (s, 9H), 0.99 (t,
J = 7.5 Hz, 3H), 0.81 (t, J = 6.9 Hz, 3H); 13C NMR
(CDCl3, 75 MHz): d: 174.8, 170.8, 158.0, 156.3, 153.7,
140.1, 135.0, 131.1, 130.1, 129.5, 124.6, 123.9, 123.7,
123.6, 122.3, 118.9, 121.6, 118.5, 117.5, 110.5, 95.1,
82.1, 79.1, 77.3, 76.0, 66.1, 55.9, 55.5, 54.6, 52.7, 52.3,
52.3, 50.2 (2C), 46.0, 45.2, 44.6, 42.8, 40.6, 34.4, 33.1,
31.6, 28.5 (3C), 27.9, 25.9, 21.1, 12.4, 8.4; ESIMS
1
CHCl3); H NMR (CDCl3, 300 MHz): d: 9.26 (s, 1H),
8.04 (s, 1H), 7.51 (d, J = 7.5 Hz, 1H), 7.12 (m, 3H),
6.59 (s, 1H), 6.20 (s, 1H), 5.87 (dd, J = 10.2, 3.9 Hz,
1H), 5.48 (d, J = 5.7 Hz, 1H), 5.42 (d, J = 10.2 Hz,
1H), 5.36 (d, J = 6.6 Hz, 1H), 5.02 (s, 1H), 3.82 (s,
3H), 3.65 (s, 3H), 3.61 (s, 3H), 3.38 (s, 1H), 2.92 (s,
3H), 2.80 (d, J = 16.2 Hz, 1H), 2.60 (s, 1H), 2.14 (s,
3H), 1.93 (q, J = 7.5 Hz, 2H), 1.46 (m, 1H), 1.23 (m,
1H), 0.99 (t, J = 7.5 Hz, 3H), 0.80 (t, J = 6.9 Hz, 3H);
13C NMR (CDCl3, 75 MHz): d: 174.9, 171.0, 158.0,
157.4, 153.7, 140.0, 135.1, 131.0, 130.0, 129.6, 124.7,
124.0, 123.7, 123.7, 122.4, 121.8, 119.0, 118.5, 117.5,
110.6, 95.3, 82.2, 76.8, 76.1, 66.1, 56.0, 55.6, 54.7, 52.8,
52.5, 52.2, 50.7, 50.2 (2C), 45.9, 45.2, 44.9, 42.8, 40.8,

Y. Shao et al. / Bioorg. Med. Chem. 15 (2007) 5061–5075
5071
(m/e) 864.4 [M+1]+. HRESIMS C50H66N5O8 [M+H]+
calcd for 864.4911, found 864.4914.
C48H61BrN5O8 [M+H]+ calcd for 916.3683, found
916.3685.
5.3.5. 3-Demethoxycarbonyl-3-(isobutyloxycarbonylami-
5.3.8. 3-Demethoxycarbonyl-3-(n-pentanyloxycarbonyl-
20
D
20
D
no)-methyl-anhydrovinblastine (12b). ½aꢁ þ 76ꢂ (c 0.06,
amino)-methyl-anhydrovinblastine (15b). ½aꢁ þ 70:4ꢂ (c
1
1
CHCl3); H NMR (CDCl3, 300 MHz) d: 9.30 (s, 1H),
8.00 (s, 1H), 7.53 (d, J = 7.5 Hz, 1H), 7.17–7.08 (m,
3H), 6.60 (s, 1H), 6.20 (s, 1H), 5.88 (dd, J = 10.2,
3.9 Hz, 1H), 5.47 (d, J = 5.4 Hz, 1H), 5.42 (d,
J = 10.2 Hz, 1H), 5.30 (d, J = 3.9 Hz, 1H), 5.04 (s,
1H), 3.84 (d, J = 6.0 Hz, 2H), 3.83 (s, 3H), 3.62 (s,
3H), 3.40 (s, 1H), 2.93 (s, 3H), 2.82 (d, J = 15.9 Hz,
1H), 2.61 (s, 1H), 2.15 (s, 3H), 1.49–1.44 (m, 1H),
1.28–1.21 (m, 1H), 1.00 (t, J = 7.2 Hz, 3H), 0.91 (d,
J = 6.0 Hz, 6H), 0.81 (t, J = 7.2 Hz, 3H); 13C NMR
(CDCl3, 75 MHz) d: 173.6, 170.6, 157.5, 156.8, 153.9,
134.6 (2C), 130.4, 129.4, 128.2, 124.6, 123.9, 123.8,
123.3, 122.6, 119.9, 119.1, 117.7, 112.9, 110.5, 94.7,
81.2, 76.8, 75.7, 70.7, 65.8, 55.7, 54.9, 54.0, 52.5,
52.3, 49.9 (2C), 48.9, 45.1, 44.6, 44.4, 42.4, 39.9,
33.7, 31.2, 29.4, 27.8, 27.5, 20.8, 19.6, 18.8 (2· C),
11.2, 8.1. ESIMS (m/z) 864.4 [M+1]+. HRESIMS
C50H66N5O8 [M+H]+ calcd for 864.4911, found
864.4915.
0.45, CHCl3); H NMR (CDCl3, 300 MHz): d: 9.27 (s,
1H), 8.00 (s, 1H), 7.54 (d, J = 7.5 Hz, 1H), 7.13 (m,
3H), 6.60 (s, 1H), 6.20 (s, 1H), 5.87 (dd, J = 10.2,
3.9 Hz, 1H), 5.48 (d, J = 6.0 Hz, 1H), 5.42 (d,
J = 10.2 Hz, 1H), 5.30 (d, J = 3.9 Hz, 1H), 5.03 (s,
1H), 4.04 (t, J = 6.0 Hz, 2H), 3.83 (s, 3H), 3.64 (s,
3H), 3.38 (s, 1H), 2.93 (s, 3H), 2.82 (d, J = 15.9 Hz,
1H), 2.61 (s, 1H), 2.16 (s, 3H), 1.60 (m, 2H), 1.46 (m,
1H), 1.26(m, 4H), 1.23 (m, 1H), 1.00 (t, J = 7.2 Hz,
3H), 0.89 (d, J = 6.6 Hz, 3H), 0.81 (t, J = 7.2 Hz, 3H);
13C NMR (CDCl3, 75 MHz): d: 174.9, 171.0, 158.1,
157.2, 153.7, 140.1, 135.1, 131.0, 130.1, 129.6, 124.7,
124.0, 123.7, 123.7, 122.4, 121.8, 119.0, 118.5, 117.5,
110.6, 95.2, 82.1, 77.2, 76.1, 66.5, 66.1, 56.0, 55.6, 54.7,
52.7, 52.5, 52.3, 50.2 (2C), 46.0, 45.3, 44.9, 42.9, 40.7,
34.5, 33.0, 31.7, 28.3, 28.0, 25.9, 22.4, 22.5, 21.1, 12.4,
10.5, 8.4; ESIMS (m/e) 878.5 [M+1]+. HRESIMS
C51H68N5O8 [M+H]+ calcd for 878.5068, found
878.5065.
5.3.6. 3-Demethoxycarbonyl-3-(2-methoxylethyloxycarbon-
5.3.9. 3-Demethoxycarbonyl-3-(cyclopropylmethyloxycar-
20
D
20
D
ylamino)-methyl-anhydrovinblastine (13b). ½aꢁ þ 59:8ꢂ (c
bonylamino)-methyl-anhydrovinblastine (16b). ½aꢁ þ 95ꢂ
1
1
0.74, CHCl3); H NMR (CDCl3, 300 MHz): d: 9.26 (s,
1H), 8.04 (s, 1H), 7.48 (d, J = 7.2 Hz, 1H), 7.11 (m,
3H), 6.49 (s, 1H), 6.19 (s, 1H), 5.87 (dd, J = 10.2,
4.5 Hz, 1H), 5.61 (d, J = 5.4 Hz, 1H), 5.50 (d,
J = 6.6 Hz, 1H), 5.41 (d, J = 10.2 Hz, 1H), 4.99 (s,
1H), 4.21 (s, 2H), 3.82 (s, 3H), 3.62 (s, 3H), 3.41 (s,
1H), 3.37 (s, 3H), 2.93 (s, 3H), 2.81 (d, J = 16.2 Hz,
1H), 2.59 (s, 1H), 2.14 (s, 3H), 1.99 (q, J = 7.5 Hz,
2H), 1.46 (m, 1H), 1.23 (m, 1H), 1.01 (t, J = 7.2 Hz,
3H), 0.77 (t, J = 6.9 Hz, 3H); 13C NMR (CDCl3,
75 MHz): d: 174.4, 171.0, 158.0, 156.7, 154.0, 137.2,
135.1, 130.8, 130.0, 129.1, 124.9, 124.2, 123.9, 123.5,
123.1, 120.6, 119.7, 118.4, 115.2, 110.7, 95.1, 81.8,
77.2, 76.1, 71.1, 66.1, 64.0, 59.0, 56.0, 55.5, 54.1, 52.7,
52.7, 50.2 (3C), 45.3, 45.1, 44.8, 42.9, 40.5, 34.3, 31.6,
31.0, 27.9, 22.2, 21.1, 11.9, 8.4; ESIMS (m/e) 866.4
[M+1]+. HRESIMS C49H64N5O9 [M+H]+ calcd for
866.4704, found 866.4701.
(c 0.18, CHCl3), H NMR (CDCl3, 300 MHz) d: 9.27
(s, 1H), 8.00 (s, 1H), 7.53 (d, J = 7.5 Hz, 1H), 7.17–
7.10 (m, 3H), 6.60 (s, 1H), 6.20 (s, 1H), 5.87 (dd,
J = 10.2, 4.2 Hz, 1H), 5.47 (d, J = 6.0 Hz, 1H), 5.42 (d,
J = 10.2 Hz, 1H), 5.36 (d, J = 4.5 Hz, 1H), 5.03 (s,
1H), 3.89 (d, J = 7.2 Hz, 2H), 3.83 (s, 3H), 3.62 (s,
3H), 3.41 (s, 1H), 2.94 (s, 3H), 2.82 (d, J = 15.9 Hz,
1H), 2.61 (s, 1H), 2.15 (s, 3H), 1.49–1.44 (m, 1H),
1.28–1.21 (m, 1H), 1.00 (t, J = 7.2 Hz, 3H), 0.81 (t,
J = 7.2 Hz, 3H), 0.51 (d, J = 4.8 Hz, 2H), 0.25 (d,
J = 4.8 Hz, 2H); 13C NMR (CDCl3, 75 MHz) d: 175.0,
171.0, 158.1, 157.1, 153.8, 140.1, 135.2, 131.2, 130.1,
129.7, 124.7, 124.1, 123.8, 123.8, 122.5, 121.8, 119.0,
118.6, 117.6, 110.7, 95.3, 82.2, 77.3, 76.1, 69.7, 66.3,
56.0, 55.7, 54.7, 52.8, 52.5, 52.4, 50.3 (2C), 46.1, 45.3,
45.0, 43.0, 40.8, 34.5, 33.1, 31.7, 28.0, 25.9, 21.2, 12.5,
10.3, 8.3, 3.2 (2· C); ESIMS (m/z) 862.4 [M+1]+. HRE-
SIMS C50H64N5O8 [M+H]+ calcd for 862.4755, found
862.4752.
5.3.7. 3-Demethoxycarbonyl-3-(bromoethyloxycarbonyl-
20
D
amino)-methyl-anhydrovinblastine (14b). ½aꢁ þ 71:4ꢂ (c
5.3.10. 3-Demethoxycarbonyl-3-(cyclobutyloxycarbonyl-
20
D
1
0.61, CHCl3); H NMR (CDCl3, 300 MHz): d: 9.32 (s,
1H), 8.02 (s, 1H), 7.51 (d, J = 7.8 Hz, 1H), 7.12 (m,
3H), 6.60 (s, 1H), 6.20 (s, 1H), 5.87 (dd, J = 10.2,
4.5 Hz, 1H), 5.47 (m, 2H), 5.42 (d, J = 10.2 Hz, 1H),
5.02 (s, 1H), 4.34 (m, 2H), 3.82 (s, 3H), 3.60 (s, 3H),
3.38 (s, 1H), 2.92 (s, 3H), 2.81 (d, J = 16.2 Hz, 1H),
2.61 (s, 1H), 2.15 (s, 3H), 1.92 (q, J = 7.5 Hz, 2H),
1.46 (m, 1H), 1.23 (m, 1H), 0.98 (t, J = 7.5 Hz, 3H),
0.80 (t, J = 6.6 Hz, 3H); 13C NMR (CDCl3, 75 MHz):
d: 174.8, 171.8, 158.0, 156.0, 153.5, 139.9, 135.0, 130.9,
129.9, 129.5, 124.6, 123.8, 123.6, 123.5, 122.3, 121.7,
118.9, 118.4, 117.4, 110.5, 95.2, 82.1, 77.0, 75.9, 65.9,
64.2, 56.0, 55.5, 54.6, 52.7, 52.4, 52.1, 50.1, 50.0, 45.9,
45.1, 44.8, 42.6, 40.8, 34.3, 32.8, 31.5, 29.7, 27.9, 25.6,
21.1, 12.3, 8.3; ESIMS (m/e) 916.3 [M+1]+. HRESIMS
amino)-methyl-anhydrovinblastine (17b). ½aꢁ þ 62:2ꢂ (c
1
1.2, CHCl3); H NMR (CDCl3, 300 MHz): d: 9.25 (s,
1H), 8.00 (s, 1H), 7.53 (d, J = 7.5 Hz, 1H), 7.13 (m,
3H), 6.60 (s, 1H), 6.20 (s, 1H), 5.87 (dd, J = 10.2,
3.9 Hz, 1H), 5.47 (d, J = 5.4 Hz, 1H), 5.42 (d,
J = 10.2 Hz, 1H), 5.30 (d, J = 3.9 Hz, 1H), 5.02 (s,
1H), 4.92 (m, 1H), 3.83 (s, 3H), 3.64 (s, 3H), 3.39 (s,
1H), 2.93 (s, 3H), 2.82 (d, J = 15.9 Hz, 1H), 2.62 (s,
1H), 2.30 (m, 2H), 2.15 (s, 3H), 2.02 (m, 2H), 1.92 (q,
J = 7.5 Hz, 2H), 1.73 (m, 1H), 1.57 (m, 1H), 1.25 (m,
1H), 1.00 (t, J = 7.5 Hz, 3H), 0.81 (t, J = 6.9 Hz, 3H);
13C NMR (CDCl3, 75 MHz): d: 174.9, 170.9, 158.0,
156.3, 153.7, 140.2, 135.1, 131.1, 130.1, 129.6, 124.7,
124.0, 123.7, 123.7, 122.4, 121.8, 119.0, 118.5, 117.6,
110.6, 95.3, 82.2, 77.1, 76.0, 68.9, 66.0, 56.0, 55.6, 54.7,

5072
Y. Shao et al. / Bioorg. Med. Chem. 15 (2007) 5061–5075
52.7, 52.5, 52.3, 50.2 (2C), 46.0, 45.3, 44.7, 42.8, 40.9,
34.4, 33.0, 31.6, 30.7, 30.5, 28.0, 25.9, 21.2, 13.3, 12.4,
8.5; ESIMS (m/e) 862.4 [M+1]+. HRESIMS
C50H64N5O8 [M+H]+ calcd for 862.4755, found
862.4754.
110.7, 95.3, 82.2, 76.8, 75.8, 65.9, 55.9, 55.3, 54.4, 52.7,
52.6, 52.5, 50.5, 50.1, 50.0, 45.3, 44.9, 42.7, 40.9, 34.1,
33.0, 31.5, 27.8 (2C), 21.1, 11.8, 8.4; ESIMS (m/e)
884.4 [M+1]+. HRESIMS C52H62N5O8 [M+H]+ calcd
for 884.4598, found 884.4594.
5.3.11. 3-Demethoxycarbonyl-3-(cyclopentyloxycarbon-
5.3.14. 3-Demethoxycarbonyl-3-(2-methoxyl-phenyloxycar-
20
D
20
D
ylamino)-methyl-anhydrovinblastine (18b). ½aꢁ þ 68:3ꢂ
bonylamino)-methyl-anhydrovinblastine (21b). ½aꢁ þ 82:0ꢂ
1
1
(c 0.64, CHCl3); H NMR (CDCl3, 300 MHz): d: 9.25
(s, 1H), 8.00 (s, 1H), 7.53 (d, J = 7.5 Hz, 1H), 7.13 (m,
3H), 6.60 (s, 1H), 6.20 (s, 1H), 5.87 (dd, J = 10.2,
3.9 Hz, 1H), 5.47 (d, J = 5.1 Hz, 1H), 5.42 (d,
J = 10.2 Hz, 1H), 5.21 (d, J = 3.9 Hz, 1H), 5.08 (m,
1H), 5.03 (s, 1H), 3.83 (s, 3H), 3.64 (s, 3H), 3.40 (s,
1H), 2.93 (s, 3H), 2.82 (d, J = 15.9 Hz, 1H), 2.60 (s,
1H), 2.40 (m, 2H), 2.16 (s, 3H), 1.92 (q, J = 7.5 Hz,
2H), 1.72 (m, 2H), 1.58 (m, 4H), 1.44 (m, 2H), 1.25
(m, 1H), 1.00 (t, J = 7.5 Hz, 3H), 0.81 (t, J = 6.9 Hz,
3H); 13C NMR (CDCl3, 75 MHz): d: 174.9, 170.9,
158.1, 156.9, 153.8, 140.1, 135.2, 131.2, 130.1, 129.6,
124.7, 124.1, 123.8, 123.8, 122.4, 121.8, 119.0, 118.6,
117.5, 110.6, 95.3, 82.2, 77.7, 77.4, 76.1, 66.3, 56.0,
55.7, 54.7, 53.6, 52.7, 52.5, 52.4, 50.3, 46.1, 45.3, 45.0,
42.9, 40.7, 34.5, 33.1, 31.7, 33.0 (2C), 28.0, 26.0, 24.0
(2C), 21.1, 12.5, 8.5; ESIMS (m/e) 876.4 [M+1]+. HRE-
SIMS C51H66N5O8 [M+H]+ calcd for 876.4911, found
876.4915.
(c 0.35, CHCl3); H NMR (CDCl3, 300 MHz): d: 9.35
(s, 1H), 8.02 (s, 1H), 7.53 (d, J = 7.8 Hz, 1H), 7.14 (m,
5H), 6.94 (d, J = 8.4 Hz, 2H), 6.63 (s, 1H), 6.23 (s,
1H), 5.89 (dd, J = 10.2, 3.9 Hz, 1H), 5.81 (d,
J = 8.1 Hz, 1H), 5.47 (m, 2H), 5.06 (s, 1H), 3.83 (s,
3H), 3.81 (s, 3H), 3.64 (s, 3H), 3.49 (s, 1H), 3.00 (s,
3H), 2.85 (d, J = 15.9 Hz, 1H), 2.65 (s, 1H), 2.18 (s,
3H), 1.92 (q, J = 7.5 Hz, 2H), 1.43 (m, 1H), 1.21 (m,
1H), 1.00 (t, J = 7.5 Hz, 3H), 0.83 (t, J = 6.9 Hz,
3H); 13C NMR (CDCl3, 75 MHz): d: 174.9, 171.0,
158.1, 154.7, 153.8, 151.9, 140.2, 140.1, 135.2, 131.2,
130.1, 129.6, 126.6, 124.8, 124.0, 123.8, 123.8, 123.5,
122.5, 121.9, 120.9, 119.1, 118.6, 117.5, 112.5, 110.7,
95.4, 82.2, 77.1, 76.2, 66.2, 56.0, 55.9, 55.7, 54.7,
52.9, 52.6, 52.4, 50.3 (2C), 46.0, 45.3, 45.1, 42.9,
40.9, 34.5, 33.1, 31.7, 28.0, 25.9, 21.2, 12.5, 8.5;
ESIMS (m/e) 914.4 [M+1]+. HRESIMS C53H64N5O9
[M+H]+ calcd for 914.4704, found 914.4701.
5.3.15. 3-Demethoxycarbonyl-3-(4-methoxyl-phenyloxycar-
20
D
5.3.12. 3-Demethoxycarbonyl-3-(cyclohexanyloxycarbon-
bonylamino)-methyl-anhydrovinblastine (22b). ½aꢁ þ 84:1ꢂ
20
D
1
ylamino)-methyl-anhydrovinblastine (19b). ½aꢁ þ 65:0ꢂ
(c 0.35, CHCl3); H NMR (CDCl3, 300 MHz): d: 9.36
(s, 1H), 8.02 (s, 1H), 7.53 (d, J = 7.5 Hz, 1H), 7.14 (m,
5H), 7.02 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 8.4 Hz, 2H),
6.63 (s, 1H), 6.23 (s, 1H), 5.89 (dd, J = 10.2, 3.9 Hz,
1H), 5.81 (d, J = 8.1 Hz, 1H), 5.47 (m, 2H), 5.07 (s,
1H), 3.83 (s, 3H), 3.79 (s, 3H), 3.64 (s, 3H), 3.46 (s,
1H), 2.98 (s, 3H), 2.85 (d, J = 15.9 Hz, 1H), 2.65 (s,
1H), 2.17 (s, 3H), 1.92 (q, J = 7.5 Hz, 2H), 1.43 (m,
1H), 1.21 (m, 1H), 1.00 (t, J = 7.5 Hz, 3H), 0.82 (t,
J = 6.9 Hz, 3H); 13C NMR (CDCl3, 75 MHz): d:
174.9, 171.0, 158.1, 157.0, 155.4, 153.7, 144.8, 140.2,
135.2, 131.1, 130.1, 129.7, 124.7, 124.0, 123.8, 123.8,
122.6 (2C), 122.5, 122.1, 119.1, 118.6, 117.5, 114.5
(2C), 110.7, 95.5, 82.7, 77.0, 76.0, 66.1, 56.0, 55.7,
54.7, 52.9, 52.7, 52.5, 52.4, 50.3 (2C), 46.0, 45.3
(2C), 42.9, 41.2, 34.5, 33.1, 31.7, 28.0, 26.0, 21.2,
12.4, 8.5; ESIMS (m/e) 914.4 [M+1]+. HRESIMS
C53H64N5O9 [M+H]+ calcd for 914.4704, found
914.4703.
1
(c 0.58, CHCl3); H NMR (CDCl3, 300 MHz): d: 9.25
(s, 1H), 8.00 (s, 1H), 7.52 (d, J = 7.5 Hz, 1H), 7.13 (m,
3H), 6.60 (s, 1H), 6.20 (s, 1H), 5.87 (dd, J = 10.2,
3.9 Hz, 1H), 5.47 (d, J = 5.1 Hz, 1H), 5.42 (d,
J = 10.2 Hz, 1H), 5.26 (d, J = 6.0 Hz, 1H), 5.03 (s,
1H), 4.62 (m, 1H), 3.83 (s, 3H), 3.63 (s, 3H), 3.40 (s,
1H), 2.93 (s, 3H), 2.82 (d, J = 15.9 Hz, 1H), 2.61 (s,
1H), 2.40 (m, 2H), 2.16 (s, 3H), 1.72 (m, 2H), 1.62 (m,
2H), 1.42 (m, 1H), 1.26 (m, 6H), 1.00 (t, J = 7.5 Hz,
3H), 0.81 (t, J = 6.9 Hz, 3H); 13C NMR (CDCl3,
75 MHz): d: 175.0, 171.0, 158.1, 156.7, 153.8, 140.1,
135.2, 131.2, 130.2, 129.7, 124.7, 124.1, 123.9, 123.8,
122.5, 121.8, 119.1, 118.6, 117.6, 110.6, 95.3, 82.2,
77.4, 76.1, 73.0, 66.3, 56.0, 55.7, 54.7, 52.8, 52.5, 52.4,
50.4 (2C), 46.1, 45.3, 45.0, 42.9, 40.7, 34.5, 33.1, 31.7
(2C), 31.7, 28.0, 25.9, 26.0, 24.0 (2C), 21.2, 12.5, 8.5;
ESIMS (m/e) 890.5 [M+1]+. HRESIMS C52H68N5O8
[M+H]+ calcd for 890.5068, found 890.5065.
5.3.13. 3-Demethoxycarbonyl-3-(phenyloxycarbonylami-
5.3.16. 3-Demethoxycarbonyl-3-(benzyloxycarbonylami-
20
D
20
D
no)-methyl-anhydrovinblastine (20b). ½aꢁ þ 85:7ꢂ (c
no)-methyl-anhydrovinblastine (23b). ½aꢁ þ 67:2ꢂ (c
1
1
0.35, CHCl3); H NMR (CDCl3, 300 MHz): d: 9.38 (s,
1H), 8.01 (s, 1H), 7.53 (d, J = 7.8 Hz, 1H), 7.35 (t,
J = 7.8 Hz, 2H), 7.14 (m, 6H), 6.63 (s, 1H), 6.23 (s,
1H), 5.89 (dd, J = 10.2, 3.9 Hz, 1H), 5.76 (d,
J = 7.8 Hz, 1H), 5.46 (m, 2H), 5.07 (s, 1H), 3.83 (s,
3H), 3.63 (s, 3H), 3.45 (s, 1H), 2.99 (s, 3H), 2.85 (d,
J = 15.9 Hz, 1H), 2.65 (s, 1H), 2.18 (s, 3H), 1.43 (m,
1H), 1.21 (m, 1H), 1.00 (t, J = 7.5 Hz, 3H), 0.82 (t,
J = 6.9 Hz, 3H); 13C NMR (CDCl3, 75 MHz): d: 174.2,
170.9, 157.8, 154.8, 153.8, 151.1, 134.9 (2C), 130.8,
129.8, 129.3 (2C), 129.3, 128.9, 125.2, 124.8, 123.9,
123.2, 123.0, 121.6 (2C), 120.7, 119.6, 118.9, 118.1,
0.48, CHCl3); H NMR (CDCl3, 300 MHz): d: 9.30 (s,
1H), 8.04 (s, 1H), 7.54 (d, J = 7.5 Hz, 1H), 7.35 (m,
5H), 7.15 (m, 3H), 6.63 (s, 1H), 6.22 (s, 1H), 5.88 (dd,
J = 10.2, 4.5 Hz, 1H), 5.48 (m, 2H), 5.42 (d,
J = 10.2 Hz, 1H), 5.12 (s, 2H), 5.05 (s, 1H), 3.84 (s,
3H), 3.63 (s, 3H), 3.40 (s, 1H), 2.93 (s, 3H), 2.82 (d,
J = 15.9 Hz, 1H), 2.63 (s, 1H), 2.14 (s, 3H), 1.95 (q,
J = 7.5 Hz, 2H), 1.46 (m, 1H), 1.28 (m, 1H), 1.01 (t,
J = 7.5 Hz, 3H), 0.83 (t, J = 6.9 Hz, 3H); 13C NMR
(CDCl3, 75 MHz): d: 174.8, 170.8, 157.9, 156.6, 153.5,
140.0, 136.7, 135.0, 131.0, 130.0, 129.5, 128.5 (2C),
128.0, 128.0 (2C), 124.6, 123.9, 123.6, 123.6, 122.3,

Y. Shao et al. / Bioorg. Med. Chem. 15 (2007) 5061–5075
5073
121.7, 118.9, 118.4, 117.5, 110.6, 95.2, 82.0, 77.0, 76.0,
66.6, 65.9, 55.9, 55.5, 54.6, 52.6, 52.4, 52.3, 50.0 (2C),
45.9, 45.1, 44.8, 42.7, 40.7, 34.4, 33.0, 31.5, 27.8, 25.9,
21.0, 12.4, 8.4; ESIMS (m/e) 898.4 [M+1]+. HRESIMS
C53H64N5O8 [M+H]+ calcd for 898.4755, found
898.4751.
171.0, 158.0, 156.7, 153.7, 147.8, 147.6, 140.0, 135.1,
131.1, 130.6, 130.0, 129.6, 124.7, 124.0, 123.7, 123.7,
122.4, 122.1, 121.8, 119.0, 118.5, 117.4, 110.6, 109.0,
108.3, 101.2, 95.3, 82.1, 77.2, 76.1, 66.7, 66.1, 56.0,
55.6, 54.7, 52.7, 52.5, 52.3, 50.2 (2C), 46.0, 45.2, 44.9,
42.8, 40.8, 34.5, 33.0, 31.7, 27.9, 25.8, 21.1, 12.4, 8.4;
ESIMS (m/e) 942.4 [M+1]+. HRESIMS C54H64N5O10
[M+H]+ calcd for 942.4653, found 942.4651.
5.3.17. 3-Demethoxycarbonyl-3-(2-methoxylbenzyloxy-
20
D
carbonylamino)-methyl-anhydrovinblastine (24b). ½aꢁ
þ
1
68:1ꢂ (c 0.46, CHCl3); H NMR (CDCl3, 300 MHz): d:
9.26 (s, 1H), 8.03 (s, 1H), 7.53 (d, J = 7.5 Hz, 1H),
7.34 (d, J = 7.5 Hz, 1H), 7.27 (d, J = 7.5 Hz, 1H), 7.13
(m, 3H), 6.93 (d, J = 7.5 Hz, 1H), 6.89 (d, J = 7.5 Hz,
1H), 6.60 (s, 1H), 6.21 (s, 1H), 5.87 (dd, J = 10.2,
3.9 Hz, 1H), 5.44 (m, 3H), 5.17 (s, 2H), 5.04 (s, 1H),
3.83 (s, 6H), 3.62 (s, 3H), 3.38 (s, 1H), 2.93 (s, 3H),
2.81 (d, J = 15.3 Hz, 1H), 2.61 (s, 1H), 2.14 (s, 3H),
1.94 (q, J = 7.5 Hz, 2H), 1.47 (m, 1H), 1.26 (m, 1H),
1.00 (t, J = 7.5 Hz, 3H), 0.82 (t, J = 6.9 Hz, 3H); 13C
NMR (CDCl3, 75 MHz): d: 174.9, 170.9, 158.0, 157.7,
156.9, 153.7, 140.1, 135.1, 131.1, 130.1, 129.6, 129.6,
129.4, 125.1, 124.6, 124.0, 123.7, 123.7, 122.4, 121.7,
120.5, 119.0, 118.5, 117.5, 110.6, 110.5, 95.2, 82.0,
77.2, 76.1, 66.1, 62.1, 56.0, 55.6, 55.5, 54.7, 52.7, 52.5,
52.3, 50.2 (2C), 46.0, 45.2, 45.0, 42.8, 40.7, 34.5, 33.0,
31.6, 28.0, 25.9, 21.1, 12.4, 8.4; ESIMS (m/e) 928.4
[M+1]+. HRESIMS C54H66N5O9 [M+H]+ calcd for
928.4860, found 928.4861.
5.3.20. 3-Demethoxycarbonyl-3-(3-chlorobenzyloxycarbon-
20
ylamino)-methyl-anhydrovinblastine (27b). ½aꢁ þ 65:1ꢂ (c
D
0.49, CHCl3); 1H NMR (CDCl3, 0 MHz): d: 9.21 (s,
1H), 7.94 (s, 1H), 7.45 (d, J = 7.5 Hz, 1H), 7.16 (m, 4H),
7.04 (m, 3H), 6.53 (s, 1H), 6.13 (s, 1H), 5.80 (dd,
J = 10.2, 3.9 Hz, 1H), 5.38 (m, 3H), 5.05 (s, 2H), 4.96 (s,
1H), 3.76 (s, 3H), 3.57 (s, 3H), 3.31 (s, 1H), 2.84 (s, 3H),
2.75 (d, J = 15.9 Hz, 1H), 2.54 (s, 1H), 2.06 (s, 3H), 1.87
(q, J = 7.5 Hz, 2H), 1.39 (m, 1H), 1.18 (m, 1H), 0.93 (t,
J = 7.5 Hz, 3H), 0.74 (t, J = 6.9 Hz, 3H); 13C NMR
(CDCl3, 75 MHz): d: 174.9, 171.0, 158.1, 156.5, 153.7,
140.0, 139.0, 135.2, 134.5, 131.1, 130.1, 129.9, 129.6,
128.3, 128.0, 126.0, 124.7, 124.0, 123.7, 123.7, 122.5,
121.9, 119.1, 118.6, 117.4, 110.7, 95.4, 82.0, 77.2, 76.1,
66.1, 65.8, 56.0, 55.7, 54.7, 52.8, 52.6, 52.3, 50.2 (2C),
46.0, 45.3, 45.0, 42.9, 40.9, 34.5, 33.0, 31.7, 28.0, 25.7,
21.1, 12.4, 8.5; ESIMS (m/e) 932.4 [M+1]+. HRESIMS
C53H63ClN5O8 [M+H]+ calcd for 932.4365, found
932.4361.
5.3.18. 3-Demethoxycarbonyl-3-(4-methoxylbenzyloxy-
5.3.21. 3-Demethoxycarbonyl-3-(4-chlorobenzyloxycarbon-
20
20
D
carbonylamino)-methyl-anhydrovinblastine (25b). ½aꢁ
þ
ylamino)-methyl-anhydrovinblastine (28b). ½aꢁ þ 65:7ꢂ (c
D
1
1
69:2ꢂ (c 0.48, CHCl3); H NMR (CDCl3, 300 MHz):
d: 9.24 (s, 1H), 8.03 (s, 1H), 7.53 (d, J = 7.5 Hz, 1H),
7.26 (d, J = 7.5 Hz, 2H), 7.13 (m, 3H), 6.87 (d,
J = 7.5 Hz, 2H), 6.61 (s, 1H), 6.20 (s, 1H), 5.87 (dd,
J = 10.5, 4.5 Hz, 1H), 5.49 (d, J = 5.4 Hz, 1H), 5.42
(m, 2H), 5.03 (s, 3H), 3.83 (s, 3H), 3.79 (s, 3H), 3.62
(s, 3H), 3.38 (s, 1H), 2.91 (s, 3H), 2.80 (d, J = 16.2 Hz,
1H), 2.61 (s, 1H), 2.13 (s, 3H), 1.94 (q, J = 7.5 Hz,
2H), 1.43 (m, 1H), 1.21 (m, 1H), 1.00 (t, J = 7.5 Hz,
3H), 0.81 (t, J = 6.9 Hz, 3H); 13C NMR (CDCl3,
75 MHz): d: 174.9, 171.0, 159.6, 158.1, 156.8, 153.7,
140.2, 135.2, 131.2, 130.1, 130.0 (2C), 129.6, 129.0,
124.7, 124.1, 123.8, 123.8, 122.4, 122.8, 119.0, 118.6,
117.6, 114.0 (2C), 110.6, 95.3, 82.1, 77.2, 76.1, 66.5,
66.1, 56.0, 55.7, 55.4, 54.7, 52.8, 52.5, 52.4, 50.2 (2C),
46.0, 45.2, 45.0, 42.9, 40.8, 34.5, 33.1, 31.7, 28.0, 26.0,
21.1, 12.5, 8.5; ESIMS (m/e) 928.4 [M+1]+. HRESIMS
C54H66N5O9 [M+H]+ calcd for 928.4860, found
928.4858.
0.47, CHCl3); H NMR (CDCl3, 300 MHz): d: 9.29 (s,
1H), 8.02 (s, 1H), 7.53 (d, J = 7.8 Hz, 1H), 7.30 (m, 4H),
7.13 (m, 3H), 6.61 (s, 1H), 6.20 (s, 1H), 5.87 (dd,
J = 10.2, 3.9 Hz, 1H), 5.45 (m, 3H), 5.06 (s, 2H), 5.03 (s,
1H), 3.83 (s, 3H), 3.62 (s, 3H), 3.37 (s, 1H), 2.90 (s, 3H),
2.81 (d, J = 15.9 Hz, 1H), 2.61 (s, 1H), 2.12 (s, 3H), 1.93
(q, J = 7.5 Hz, 2H), 1.47 (m, 1H), 1.24 (m, 1H), 1.00 (t,
J = 7.5 Hz, 3H), 0.81 (t, J = 6.9 Hz, 3H); 13C NMR
(CDCl3, 75 MHz): d: 174.9, 171.0, 158.1, 156.5, 153.7,
140.2, 135.5, 135.2, 134.0, 131.2, 130.1, 129.7, 129.6 (2C),
128.8 (2C), 124.7, 124.0, 123.8, 123.7, 122.5, 122.0, 119.1,
118.6, 117.6, 110.7, 95.4, 82.3, 77.2, 76.1, 66.1, 65.9, 56.1,
55.7, 54.7, 52.8, 52.6, 52.4, 50.3 (2C), 46.1, 45.3, 45.0,
42.9, 40.9, 34.5, 33.1, 31.7, 28.0, 26.0, 21.2, 12.5, 8.5;
ESIMS (m/e) 932.4 [M+1]+. HRESIMS C53H63ClN5O8
[M+H]+ calcd for 932.4365, found 932.4362.
5.3.22. 3-Demethoxycarbonyl-3-(2-nitro-benzyloxycarbon-
20
D
ylamino)-methyl-anhydrovinblastine (29b). ½aꢁ þ 66:4ꢂ (c
1
0.48, CHCl3); H NMR (CDCl3, 300 MHz): d: 9.35 (s,
1H), 8.03 (s, 1H), 8.07 (d, J = 8.1 Hz, 1H), 7.60 (m, 2H),
7.52 (d, J = 7.5 Hz, 1H), 7.46 (m, 1H), 7.13 (m, 3H),
6.62 (s, 1H), 6.22 (s, 1H), 5.88 (dd, J = 10.2, 3.9 Hz, 1H),
5.51 (m, 3H), 5.51 (s, 2H), 5.05 (s, 1H), 3.83 (s, 3H), 3.64
(s, 3H), 3.41 (s, 1H), 2.93 (s, 3H), 2.83 (d, J = 16.2 Hz,
1H), 2.63 (s, 1H), 2.14 (s, 3H), 1.93 (q, J = 7.5 Hz, 2H),
1.47 (m, 1H), 1.24 (m, 1H), 1.00 (t, J = 7.5 Hz, 3H), 0.82
(t, J = 6.9 Hz, 3H); 13C NMR (CDCl3, 75 MHz): d:
174.9, 170.9, 158.1, 156.0, 153.6, 147.2, 140.1, 135.1,
133.7, 133.7, 131.1, 130.0, 129.6, 128.8, 128.6, 125.0,
124.7, 124.0, 123.7, 123.7, 122.4, 122.0, 119.0, 118.6,
117.5, 110.6, 95.3, 82.3, 77.1, 76.0, 66.1, 63.3, 56.1, 55.6,
5.3.19. 3-Demethoxycarbonyl-3-(piperonyloxycarbonyl-
20
D
amino)-methyl-anhydrovinblastine (26b). ½aꢁ þ 63:5ꢂ (c
1
0.38, CHCl3); H NMR (CDCl3, 300 MHz): d: 9.26 (s,
1H), 8.03 (s, 1H), 7.52 (d, J = 7.5 Hz, 1H), 7.13 (m,
3H), 6.80 (m, 3H), 6.60 (s, 1H), 6.21 (s, 1H), 5.94 (s,
2H), 5.87 (dd, J = 10.5, 4.5 Hz, 1H), 5.49 (d,
J = 6.0 Hz, 1H), 5.42 (d, J = 10.5 Hz), 5.03 (s, 3H),
5.00 (s, 2H), 3.83 (s, 3H), 3.62 (s, 3H), 3.35 (s, 1H),
2.91 (s, 3H), 2.81 (d, J = 15.9 Hz, 1H), 2.61 (s, 1H),
2.13 (s, 3H), 1.94 (q, J = 7.5 Hz, 2H), 1.43 (m, 1H),
1.21 (m, 1H), 1.00 (t, J = 7.5 Hz, 3H), 0.81 (t,
J = 6.9 Hz, 3H); 13C NMR (CDCl3, 75 MHz): d: 174.9,

5074
Y. Shao et al. / Bioorg. Med. Chem. 15 (2007) 5061–5075
54.7, 52.8, 52.5, 52.4, 50.2 (2C), 46.0, 45.3, 45.0, 42.8, 40.9,
34.5, 33.1, 31.6, 28.0, 26.0, 21.1, 12.4, 8.4; ESIMS (m/e)
943.4 [M+1]+. HRESIMS C53H63N6O10 [M+H]+ calcd
for 943.4605, found 943.4607.
5.4. In vitro cytotoxicity assay
Ditartrate of all compounds was used in bioassays.
AVLB and NVB were purchased from Shanghai Kan-
g’ai Biological Product Co., Ltd. A human non-small
cell lung cancer cell line (A549) and a human cervix epi-
thelial adenocarcinoma cell line (HeLa), obtained from
American Type Culture Collection (Rockville, MD),
were used for the cytotoxicity assay in vitro by sulforho-
damine B (SRB) assay14. Briefly, the cells were seeded at
6000 cells/well in 96-well plates (Falcon, CA, USA) and
allowed to attach overnight. The cells were treated in
triplicate with grade concentrations of compounds at
37 ꢁC for 72 h. Then, they were fixed with 10% trichloro-
acetic acid and incubated for 60 min at 4 ꢁC. Then, the
plates were washed and dried. SRB solution (0.4% w/v
in 1% acetic acid) was added and the culture was incu-
bated for an additional 15 min. After the plates were
washed and dried, bound stain was solubilized with Tris
buffer, and the optical densities were read on the plate
reader (model VERSA Max, Molecular Devices) at
515 nm (A515). The results were expressed as IC50 (the
compound concentration required for 50% growth inhi-
bition of tumor cells), which was calculated by the Logit
method. The mean IC50 was determined from the results
of three independent tests.
5.3.23. 3-Demethoxycarbonyl-3-(4-nitro-benzyloxycarbon-
20
D
ylamino)-methyl-anhydrovinblastine (30b). ½aꢁ þ 66:7ꢂ
1
(c 0.49, CHCl3); H NMR (CDCl3, 300 MHz): d: 9.36
(s, 1H), 8.19 (d, J = 8.7 Hz, 2H), 8.03 (s, 1H), 7.52 (d,
J = 7.5 Hz, 1H), 7.52 (d, J = 8.7 Hz, 2H), 7.13 (m,
3H), 6.61 (s, 1H), 6.21 (s, 1H), 5.88 (dd, J = 10.2,
3.9 Hz, 1H), 5.49 (m, 3H), 5.19 (s, 2H), 5.03 (s, 1H),
3.83 (s, 3H), 3.62 (s, 3H), 3.38 (s, 1H), 2.90 (s, 3H),
2.83 (d, J = 16.2 Hz, 1H), 2.63 (s, 1H), 2.13 (s, 3H),
1.93 (q, J = 7.5 Hz, 2H), 1.47 (m, 1H), 1.24 (m, 1H),
1.00 (t, J = 7.5 Hz, 3H), 0.81 (t, J = 6.9 Hz, 3H); 13C
NMR (CDCl3, 75 MHz): d: 174.9, 170.9, 158.1, 156.2,
153.6, 147.7, 144.4, 140.1, 135.2, 131.0, 130.0, 129.6,
128.2 (2C), 124.7, 123.9, 123.8 (3C), 123.7, 122.5,
122.1, 119.0, 118.5, 117.6, 110.6, 95.4, 82.4, 77.1, 76.0,
66.1, 65.2, 56.1, 55.7, 54.7, 52.8, 52.5, 52.3, 50.2 (2C),
46.0, 45.3, 45.1, 42.8, 41.0, 34.5, 33.1, 31.7, 28.0, 25.9,
21.1, 12.4, 8.4; ESIMS (m/e) 943.4 [M+1]+. HRESIMS
C53H63N6O10 [M+H]+ calcd for 943.4605, found
943.4606.
5.3.24. 3-Demethoxycarbonyl-3-(20-oxo-3,50-spirooxazoli-
20
D
dino)-methyl-anhydrovinblastine (31b). ½aꢁ ꢀ 30:7ꢂ (c
5.5. In vivo antitumor assay of 8b
1
0.43, CHCl3); H NMR (CDCl3, 300 MHz): d: 8.43 (s,
1H), 7.51 (d, J = 7.2 Hz, 1H), 7.12 (m, 3H), 6.63 (s,
1H), 6.03 (s, 1H), 5.80 (s, 1H), 5.75 (dd, J = 10.2,
3.9 Hz, 1H), 5.46 (d, J = 5.7 Hz, 1H), 5.27 (d,
J = 10.2 Hz, 1H), 5.23 (s, 1H), 3.82 (s, 3H), 3.79 (s, 1H),
3.63 (s, 3H), 3.12 (s, 3H), 2.39 (s, 1H), 2.03 (s, 3H), 0.99
(t, J = 7.5 Hz, 3H), 0.90 (t, J = 6.9 Hz, 3H); 13C NMR
(CDCl3, 75 MHz): d: 175.4, 171.3, 158.4, 158.0, 150.4,
140.2, 135.3, 131.4, 129.2, 127.9, 126.8, 126.8, 124.3,
122.5, 122.1, 118.9, 118.8, 118.3, 116.9, 110.7, 90.9,
87.2, 74.8 (2C), 62.7, 56.2, 55.9, 54.3, 53.5, 52.5, 52.4,
52.2, 51.5, 46.2, 45.5, 44.9, 44.4, 34.8, 34.0, 33.6, 28.9,
27.9, 25.9, 21.3, 12.4, 9.2; ESIMS (m/e) 790.4 [M+1]+.
HRESIMS C46H56N5O7 [M+H]+ calcd for 790.4179,
found 790.4180.
Female 7-week-old specific pathogen free (SPF) BALB/
cA-nude mice (weight, 18–22 g) were obtained from
Shanghai Laboratory Animal Center, Chinese Academy
of Sciences. A549 cells (2.5 · 106) were subcutaneously
implanted into the axilla region of mice. After tumor vol-
ume reached 100–300 mm3, mice were intravenously gi-
ven 8b on day 1 at the doses of 1.5 mg/kg, 3 mg/kg, and
6 mg/kg, and 1d on days 1 and 4 at the dose of 10 mg/
kg. Tumor volume and mice weight were measured three
times a week. Tumor growth inhibition was calculated as
[(Average relative tumor volume of control
group ꢀ Average relative tumor volume of test group)/
Average relative tumor volume of control group] · 100%
on day 12 after dosing. Data were presented as
means SD, and significance was assessed with Student’s
t test. Difference was considered significant at p < 0.01.
5.3.25. 3-Demethoxycarbonyl-3-(isobutylcarbamoyloxy)-
20
D
methyl-anhydrovinblastine (32b). ½aꢁ þ 68:0ꢂ (c 0.38,
1
CHCl3); H NMR (CDCl3, 300 MHz): d: 9.29 (s, 1H),
8.03 (s, 1H), 7.53 (d, J = 7.5 Hz, 1H), 7.14 (m, 3H), 6.60
(s, 1H), 6.18 (s, 1H), 5.88 (dd, J = 10.2, 3.9 Hz, 1H),
5.51 (s, 1H), 5.42 (d, J = 10.2 Hz, 1H), 5.06 (s, 1H),
4.90(m, 1H), 4.10 (s, 2H), 3.84 (s, 3H), 3.63 (s, 3H), 3.54
(s, 1H), 2.92 (s, 3H), 2.81 (d, J = 15.6 Hz, 1H), 2.61 (s,
1H), 2.19 (s, 3H), 1.94 (q, J = 7.5 Hz, 2H), 1.46 (m,
1H), 1.23 (m, 1H), 1.03 (t, J = 7.5 Hz, 3H), 0.92 (d,
J = 7.2 Hz, 3H), 0.82 (t, J = 6.9 Hz, 3H); 13C NMR
(CDCl3, 75 MHz): d: 174.9, 171.0, 158.1, 156.3, 153.6,
140.2, 135.1, 131.2, 129.9, 129.6, 124.8, 124.0, 123.9,
123.8, 122.4, 121.5, 119.0, 118.5, 117.5, 110.6, 94.8,
81.8, 76.9, 76.3, 67.0, 66.4, 56.0, 55.6, 54.6, 52.6, 52.5,
52.4, 50.5, 50.2, 46.1, 45.2, 42.7, 40.9, 40.0, 34.5, 33.0,
32.1, 31.6, 27.9, 25.9, 21.1, 20.0, 13.8, 12.4, 8.4; ESIMS
(m/e) 864.3 [M+1]+. HRESIMS C50H66N5O8 [M+H]+
calcd for 864.4911, found 864.4913.
Acknowledgments
We gratefully acknowledge financial support from
Chinese
National
High-tech
R&D
Program
(2006AA020602) and Chinese Academy of Sciences
(O6G8031014).
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