In search of a cytostatic agent derived from the alkaloid lycorine: Synthesis and growth inhibitory properties of lycorine derivatives

Article abstract of DOI:10.1016/j.bmc.2011.09.051

As a continuation of our studies aimed at the development of a new cytostatic agent derived from an Amaryllidaceae alkaloid lycorine, we synthesized 32 analogues of this natural product. This set of synthetic analogues included compounds incorporating sel

Full text of DOI:10.1016/j.bmc.2011.09.051

Bioorganic & Medicinal Chemistry 19 (2011) 7252–7261
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Bioorganic & Medicinal Chemistry
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In search of a cytostatic agent derived from the alkaloid lycorine: Synthesis
and growth inhibitory properties of lycorine derivatives
Nikolai M. Evdokimov a, , Delphine Lamoral-Theys b, , Véronique Mathieu c, Anna Andolfi d,
Liliya V. Frolova a, Stephen C. Pelly e, Willem A.L. van Otterlo e, Igor V. Magedov a, Robert Kiss c,
Antonio Evidente d, Alexander Kornienko a,
⇑
a Department of Chemistry, New Mexico Institute of Mining and Technology, Socorro, NM 87801, USA
b Laboratoire de Chimie Analytique, Toxicologie et Chimie Physique Appliquée, Brussels, Belgium
c Laboratoire de Toxicologie, Faculté de Pharmacie, Université Libre de Bruxelles, Brussels, Belgium
d Dipartimento di Scienze, del Suolo, della Pianta, dell’Ambiente e delle Produzioni Animali, Università di Napoli Federico II, Via Università 100, 80055 Portici, Italy
e Department of Chemistry and Polymer Sciences, Stellenbosch University, Stellenbosch, Western Cape, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 July 2011
Revised 27 August 2011
Accepted 6 September 2011
Available online 2 October 2011
As a continuation of our studies aimed at the development of a new cytostatic agent derived from an
Amaryllidaceae alkaloid lycorine, we synthesized 32 analogues of this natural product. This set of syn-
thetic analogues included compounds incorporating selective derivatization of the C1 versus C2 hydroxyl
groups, aromatized ring C, lactamized N6 nitrogen, dihydroxylated C3–C3a olefin functionality, trans-
posed olefin from C3–C3a to C2–C3 or C3a–C4, and C1 long-chain fatty esters. All synthesized compounds
were evaluated for antiproliferative activities in vitro in a panel of tumor cell lines including those exhib-
iting resistance to proapoptotic stimuli and representing solid cancers associated with dismal prognoses,
such as melanoma, glioblastoma, and non-small-cell lung cancer. Most active analogues were not dis-
criminatory between cancer cells displaying resistance or sensitivity to apoptosis, indicating that these
compounds are thus able to overcome the intrinsic resistance of cancer cells to pro-apoptotic stimuli.
1,2-Di-O-allyllycorine was identified as a lycorine analogue, which is 100 times more potent against a
U373 human glioblastoma model than the parent natural product. Furthermore, a number of synthetic
analogues were identified as promising for the forthcoming in vivo studies.
Keywords:
Cancer
Apoptosis resistance
Lycorine
Melanoma
Glioblastoma
Alkaloid
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
anticancer properties have been evaluated in vitro and in vivo in
various preclinical models of human cancers.6 These promising
Glioma, melanoma, oesophageal, non-small-cell lung, and a
number of other types of cancer known to be associated with dis-
mal prognoses, all have the characteristic of being intrinsically
resistant to pro-apoptotic stimuli.1–4 Moreover, more than 90% of
cancer patients die from tumor metastases, which are incompetent
in initiating the apoptotic program as well. Nonetheless, a large
majority of therapeutic agents used by oncologists to treat cancer
patients are pro-apoptotic.1,2 Thus, the rise in incidence of gliomas
and melanomas has not been paralleled by improved therapeutic
options over the years. New types of drugs are, therefore, urgently
needed to combat cancers that are resistant to proapoptotic stimuli
and thus poorly responsive to current therapies.
biological results have led to some exploration of structure–
activity relationships (SAR) through isolation of lycorine’s natural
congeners and synthesis of its analogues.6e,f,h,7 Recently, we re-
ported biochemical experiments providing a mechanistic insight
into lycorine’s antiproliferative effects.7a We showed that at thera-
peutic concentrations, lycorine does not induce apoptosis in cancer
cells, but rather exhibits cytostatic effects through the targeting of
the actin cytoskeleton. This mode of action accounts for its prom-
ising activity in vitro against a panel of cancer cell lines, resistant to
proapoptotic stimuli, as well as in vivo in a mouse melanoma mod-
el. Additionally, we provided considerable evidence supporting the
eEF1A elongation factor as a likely intracellular target for the struc-
turally related Amaryllidaceae isocarbostyrils, identifying this pro-
tein as a potential target for lycorine as well.8 These results make
lycorine an excellent lead for the generation of compounds able
to combat cancers, which are naturally resistant to pro-apoptotic
stimuli, such as glioblastoma, melanoma, non-small-cell-lung and
metastatic tumors, among others. In this article, we describe the
synthesis of 32 lycorine analogues and their evaluation against a
Lycorine, a phenanthridine alkaloid isolated from various spe-
cies of the Amaryllidaceae plant family, has attracted considerable
attention due to its promising biological activities.5 Specifically, its
⇑
Corresponding author. Tel.: +1 575 835 5884; fax: +1 575 835 5364.
E-mail address: akornien@nmt.edu (A. Kornienko).
These two authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.051

N. M. Evdokimov et al. / Bioorg. Med. Chem. 19 (2011) 7252–7261
7253
panel of cancer cell lines diversely representing these challenging
types of human malignancy.
of different crystalline forms of 1), we were able to prepare C2-
pivaloate 15, C2-tri-iso-propylsilyl ether 16 and C2-benzoate 19
with exclusive regioselectivity. We also found that the C1- and
C2-hydroxyls could be differentiated by a controlled hydrolysis
of dibenzoate 17 to give corresponding mono-benzoates 18 and
19 (Scheme 4).
2. Results and discussion
2.1. Chemistry
Utilizing the facile selective protection of the C2-hydroxyl as
the tri-iso-propylsilyl ether, a number of C1-derivatives were ob-
tained. To this end, lycorine (1) was treated with i-Pr3SiCl and pyr-
idine in DMF and the crude silyl ether was acylated or alkylated.
Without purification this material was then desilylated to produce
palmitate 22, acetate 23, stearate 24, oleate 25, docosahexaenoate
26, and benzyl ether 27 (Fig. 1). Intermediate silyl ethers 20 and 21
were also isolated and purified for biological testing.
In an attempt to prepare aromatic lycorine derivatives, we
found that a simple treatment of lactam 7 with thionyl chloride
in pyridine produced indoline lactam 28 (Scheme 5), which was
previously synthesized by total synthesis efforts and converted to
indole lactam 29 by oxidation with DDQ.12 This oxidation repro-
duced well in our hands and was applied to prepare indole lactam
30 with equal success.
Lastly, conversion of the C1- and C2-hydroxyl functionality into
lithium and sodium bis-alkoxides allowed us to synthesize di-Boc
carbonate 31 and diallyl ether 32 without the competing quatern-
ization of the basic nitrogen (Scheme 6). The attempted Claisen
rearrangement using ethyl orthoacetate and propionic acid did
not lead to the desired product containing a new quaternary center
at C3a and a transposed olefin at C2–C3. Instead, dipropionate 33
was isolated from this reaction mixture as the only product. We
wondered whether this unsuccessful outcome was caused by a
competing formation of the cyclic orthoester at the C1- and C2-hy-
droxyl functionality. Therefore, this reaction was performed on
C1-protected acetate 23 and, gratifyingly gave the desired
rearranged product 34 under slightly modified conditions in a
reasonable yield of 38%.
Because lycorine is poorly soluble in most organic solvents, the
initial derivatization efforts centered on the use of its diacetate 2
(Scheme 1). For the purposes of a systematic SAR study, it was
decided to prepare some of the previously reported lycorine ana-
logues together with the new derivatives. Thus, diol 3 had been syn-
thesized previously on two occasions by a low-yielding reaction of
diacetate 2 with KMnO4.5b,9 In our hands, the synthetically useful
yield of 3 was achieved through the use of the OsO4/NMO system.
The aromatized derivative 4, obtained as a minor byproduct, was
further transformed to phenol 5. The known reaction of 2 with
PhI(OAc)2 provided lactam 6,10 which was hydrolyzed to lactam diol
7 and additionally converted to a new derivative, thiolactam 8.
Furthermore, diol 3 was used to synthesize new polyhydroxylated
derivatives 9, 10, and 13 through the application of basic
hydrolysis (9), LiAlH4 reduction (10) and secondary hydroxyl acety-
lation/elimination/hydrolysis sequence9 (11?12?13), respectively
(Scheme 2).
Next, we investigated various methods to selectively function-
alize either the C1- or C2-hydroxyl groups. We expected the
C2-hydroxyl to be more reactive both due to its allylic nature
and steric accessibility. Indeed, it was previously reported that
the C2-position could be selectively oxidized with either Jones
reagent5b or the DCC/DMSO system,11 albeit in rather low yields.
We found that the Dess–Martin reagent afforded the desired
ketone 14 in a useful 66% yield and exclusive C2-regioselectivity
(Scheme 3). In addition, although selective silylation of the
C2-hydroxyl with t-BuMe2SiCl described by McNulty et al.5a did
not work in our hands (possibly due to differences in solubility
OH
OH
OAc
N
HO
OH
OH
HO
AcO
2
3
1
3a
4
12b
O
O
O
O
O
O
N
7
N
1
O
S
8
9
Ac2O, py
99%
OAc
LiOH
P2S5
HMDSO
93%
OAc
88%
OAc
THF/H2O
OAc
N
AcO
AcO
AcO
OH
OH
OsO4, NMO
acetone
PhI(OAc)2, TBAI
MeCN, 78%
O
O
O
O
O
O
N
N
N
O
O
O
2
O
O
6
3 (53%)
4 (13%)
LiOH
THF/H2O
95%
OH
LiAlH4
LiOH
66%
93%
OH
THF
THF/H2O
OH
HO
HO
OH
OH
O
O
O
O
O
O
N
N
N
O
O
10
5
7
Scheme 1. Synthesis of known and new derivatives of lycorine starting with diacetate 2.

7254
N. M. Evdokimov et al. / Bioorg. Med. Chem. 19 (2011) 7252–7261
OAc
OAc
N
OH
N
AcO
OAc
OH
AcO
OAc
HO
OH
LiOH
SOCl2, py
72%
Ac2O, py
90%
THF/H2O
3
O
O
O
O
O
O
90%
N
O
O
O
11
12
13
Scheme 2. Synthesis of a new polyhydroxylated analogue 13.
O
cancer (NSCLC),13b,16 and human SKMEL-28 melanoma17] as well
as apoptosis-sensitive tumor models (human Hs683 anaplastic
oligodendroglioma,13b,15a human LoVo colon cancer15c,d and
mouse B16F10 melanoma).17,18 Analysis of these data reveals that
lycorine, as well as most of its active derivatives, does not dis-
criminate between the cancer cell lines based on the apoptosis
sensitivity criterion and displays comparable potencies in both
cell types, further corroborating our conclusion that apoptosis
induction is not the primary mechanism responsible for antipro-
liferative activity in this series of compounds, at least in solid
cancers (Table 1, Fig. 2).7a Although the large variations in cancer
cell line sensitivities preclude reliable statistical analyses to be
performed, compound 23 stands out as uniformly potent in inhib-
iting the growth of apoptosis-resistant cancer cells (Fig. 2).
The data also indicate the absolute requirement for the pres-
ence of a basic nitrogen at N6, as the compounds oxidized at C7,
and thus containing amidic nitrogen at this position, are inactive
(3, 6, 7, 9, 11, 12, and 13). In the same vein, thiolactam 8 also dis-
plays reduced activity. Exceptions to this rule constitute com-
pounds with an aromatic ring C (4, 5, 28, 29, and 30) and the
resultant planarity of the pentacyclic skeleton.
O
O
N
O
N
HO
HO
O
O
O
O
PivCl
py
15
1 eq BzCl
py
19
73%
68%
OH
HO
O
N
O
DMP
PhCH3
1
i-Pr3SiCl
py, DMF
79%
66%
O
OSi(i-Pr)3
HO
HO
O
O
O
O
N
N
The sterically bulky substituents at the C1- and C2-positions
appear to be tolerated well, provided they can be removed by
intracellular hydrolysis. Such compounds include dipropionate
33, dicarbonate 31, and the benzoate substituted analogues (17,
18, and 19). Of interest is the comparison of ester 17 with ether
27, where the significantly higher potency of the hydrolyzable
analogue 17 leads to a proposal that these large hydrophobic ele-
ments are not part of the pharmacophore but only assist in cell
penetration. This is also consistent with the observation that tetra-
ol 10, lying at the other end of the polarity spectrum, is inactive. A
possible exception is represented by moderately active C2-silyl
ethers (16, 20, and 21), which could be resistant to intracellular
hydrolysis.
14
16
Scheme 3. Selective functionalization of the C1 and C2-hydroxyl functionality.
O
O
O
3 eq BzCl
O
py
1
83%
O
O
N
17
The idea behind synthesizing the long-chain fatty acid deriva-
tives was to make these molecules selectively toxic to tumors as
opposed to normal tissues. Certain fatty acids have shown
in vitro and in vivo growth inhibitory effect against cancer cells19
and, more importantly, many natural fatty acids are selectively ta-
ken up by tumors, presumably for use as biochemical precursors
and energy sources.20 This phenomenon has been utilized by Brad-
ley et al. to prepare a paclitaxel–docosahexanoic acid conjugate,
which showed enhanced antitumor activity in mice compared to
that of paclitaxel itself.21 Furthermore, Ahn and co-workers have
synthesized a series of saturated and unsaturated fatty acid esters
of 40-demethyldeoxypodophyllotoxin and shown that many of
such derivatives possessed enhanced anticancer activities.22
Although analysis of the GI50 values associated with palmitate
22, stearate 24, oleate 25, and docosahexaenoate 26 reveals that
these compounds do not display elevated potencies compared with
those of lycorine (1) in vitro, the low micromolar potencies associ-
ated with these compounds and their chemical stability in the
range of pH 5–7.4 for at least 3 days are encouraging and bode well
for the forthcoming in vivo tests where their propensity for selec-
tive uptake into tumors can be properly evaluated.
LiOH
MeOH
O
O
O
N
OH
N
HO
O
O
O
O
O
18 (33%)
19 (56%)
Scheme 4. Preparation of C1- and C2-benzoates.
2.2. In vitro growth inhibitory properties
The synthesized compounds were evaluated for in vitro
growth inhibition using the MTT colorimetric assay13 against a
panel of eight cancer cell lines including those resistant to proa-
poptotic stimuli [human U373 and T98G glioblastoma (GBM, from
astroglial origin14),13b,15a,b human A549 non-small-cell-lung

N. M. Evdokimov et al. / Bioorg. Med. Chem. 19 (2011) 7252–7261
7255
OH
1. i-Pr3SiCl, py, DMF
OH
HO
RO
2. DCC, DMAP, RCO2H
or
O
O
O
O
BnBr, NaH, DMF
N
N
3. TBAF, THF
1
22−27
O
O
OH
N
OSi(i-Pr)3
N
OSi(i-Pr)3
OH
AcO
AcO
O
O
14
14
O
O
O
O
O
O
O
N
N
O
22 49%
23 51%
20 56%
21 63%
O
O
O
O
OH
H
OH
N
OH
N
OH
O
O
O
16
11
7
6
O
O
O
O
H
N
O
N
O
O
O
25 41%
26 51%
27 30%
24 53%
Figure 1. Lycorine derivatives obtained through selective protection of the C2-hydroxyl [% yields are from lycorine (1)].
O
OH
N
O
O
O
N
HO
O
N
O
O
O
O
O
N
O
O
O
O
O
O
O
29
Boc2O
BuLi
THF
allylBr
NaH
O
SOCl2
py
32
DDQ
PhH
31
7
DMF
92%
35%
98%
82%
1
O
O
CH3CH2CO2H
PhEt, reflux
MeC(OEt)3
N
O
O
28
O
2
HO
3
OEt
O
OAc
N
OAc
N
O
O
O
3a
O
O
O
N
DDQ, PhH
95%
N
O
O
O
O
33
0 %
O
30
O
4
OH
N
2
AcO
MeC(OEt)3, TsOH
Pd(OAc)2, PhEt
3
OEt
AcO
O
O
3a
Scheme 5. Synthesis of aromatic derivatives.
O
O
O
reflux, 38%
N
34
23
Importantly, diallyllycorine 32 is equipotent to lycorine (1)
across the entire panel of cell lines and as much as 100 times more
potent against the apoptosis-resistant U373 glioblastoma. Because
it is unlikely that the allyl ether functionality is removed by intra-
cellular hydrolysis, this structural element must be part of the lyc-
orine pharmacophore and a successful computer-based theoretical
model must be able to account for this result.
Scheme 6. Synthesis of dicarbonate and diether derivatives and C3a-quaterniza-
tion/olefin transposition using Claisen rearrangement.
hydrophilic lycorine analogues and it provides useful guidelines for
future analogue synthesis.
The calculated logP values for each synthesized analogue are
also listed in the Table 1. Although a direct correlation between
the lipophilicity and anticancer activity cannot be easily seen, it
is noteworthy that compounds with clogP values lower than those
of lycorine are all inactive (see 3, 7, 9, 10, 11, 13). It is likely that
this observation reflects the difficulty of cell penetration for highly
3. Conclusions
The synthesis and evaluation of 32 analogues of lycorine re-
vealed structural elements necessary for antiproliferative activity
in this series of compounds. 1,2-Di-O-allyllycorine was identified

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N. M. Evdokimov et al. / Bioorg. Med. Chem. 19 (2011) 7252–7261
Table 1
In vitro growth inhibitory effects of lycorine derivatives
Lycorine derivatives
cLogP
GI50 in vitro values (
lM)
Glioma
Carcinoma
A549
Melanoma
SKMEL-28
Mean SEM
Hs683
T98G
U373
PC3
MCF7
B16
Lycorine (1)
3
4
5
6
7
8
9
0.5
0.3
2.5
2.4
1.1
ꢀ0.3
2.6
ꢀ1.8
ꢀ0.9
0.1
0.9
ꢀ1.0
0.4
2.7
3.4
5.4
3.0
3.0
9.4
4.1
8.2
1.2
8.8
9.3
6.9
2.8
2.9
3.3
2.8
3.8
3.1
2.7
2.2
0.9
3
3
0.9
4
4
4
2
3
>98
>37 10
>46
>66 11
>100
>53
>99
>98
1
a
a
a
a
a
a
a
86
20
38
a
a
20
40
29
41
21
49
23
44
33
50
48
9
9
a
a
49
32
39
36
99
75
a
a
a
a
a
a
a
a
a
a
a
a
a
a
52
57
46
57
35
47
30
8
a
a
a
a
a
a
96
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
a
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
83
a
a
a
a
>100
>98
>100
83
a
3
38
12
11
54
32
70
3
6
7
2
16
0.8
3
34
24
96
18
4
45
9
13
1
0.6
1
13
15
24
4
8
4
8
50
4
4
7
92
12
18
11
5
34
20
26
29
3
35
36
24
20
5
23
25
27
32
4
38
30
21
74
16
27
7
16
5
22
24
8
21
38
30
28
43
23
21
18
57
8
>42 13
15
22
10
4
10
26
29
4
60
38
34
35
3
15
19
25
11
4
2
7
5
15
54
32
70
9
18
26
3
23
14
15
67
24
96
18
3
6
36 10
21
36
27
11
24
14
20
80
14
19
14
9
21
24
26
9
24
17
16
>70
20
>62 14
14
9
3
19
>97
5
4
3
5
26
22
19
97
34
30
3
4
3
25
a
8
38
33
4
a
a
27
5
4
34
5
4
4
29
79
21
22
1
21
20
6
3
3
1
5
0.2
2
4
0.03
4
8
0.6
3
4
41
22
43
6
a
a
a
a
a
a
a
a
>100 lM
resistance to proapoptotic stimuli, this finding further corroborates
our conclusion that apoptosis induction is not the primary mecha-
nism responsible for the antiproliferative activity in this series of
compounds and bodes well for the discovery of a lycorine-derived
agent able to combat such malignancies as glioblastoma, mela-
noma, non-small-cell-lung, metastatic cancers, among others. The
synthetic chemistry findings and the new SAR insights described
in this article are a valuable step toward this ambitious goal.
4. Experimental
4.1. Chemistry
All reagents, solvents and catalysts were purchased from com-
mercial sources (Acros Organics and Sigma–Aldrich) and used
without purification. CH2Cl2 was distilled from CaH2 and kept un-
der argon; THF and toluene were distilled from sodium/benzophe-
none prior to use. Lycorine was isolated from dried bulbs of
Sterbergia lutea Ker Gawl according to a published procedure23
and its purity was confirmed by NMR and HRMS. All reactions were
performed in oven-dried flasks under nitrogen or argon and mon-
Figure 2. Illustration of the in vitro growth inhibitory effects of lycorine and 17 of
its derivatives in four cancer cell lines that are sensitive to proapoptotic stimuli (the
hatched bars) versus four cancer cell lines that display resistance to pro-apoptotic
stimuli (the black bars). The data are presented as mean SEM values. We included
in the present analysis all the compounds displaying GI50 values <100
the eight cancer cell lines under study (see Table 1).
lM in each of
itored by thin layer chromatography on TLC precoated (250 lm)
silica gel 60 F254 glass-backed plates (EMD Chemicals Inc.). Visu-
alization was accomplished with UV light. Flash column chroma-
tography was performed on silica gel (32–63 lm, 60 Å pore size).
as a lycorine analogue, which is 100 times more potent against an
U373 human glioblastoma model in vitro. Because active lycorine
derivatives are equally potent toward the cell lines exhibiting
1H and 13C NMR spectra were recorded on Jeol Eclipse 300 or Bru-
ker Avance III 400 spectrometers. Chemical shifts (d) are reported
in ppm relative to the TMS internal standard. HRMS analyses were

N. M. Evdokimov et al. / Bioorg. Med. Chem. 19 (2011) 7252–7261
7257
performed at the Mass Spectrometry Facility, University of New
Mexico. Samples were run on LCT Premier TOF mass spec.
4.1.3.4. (1S,2R,3R,3a1S,12bS)-1,2,3-Trihydroxy-2,3,5,12b-tetrahy-
dro-1H-[1,3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridin-
7(3a1H)-one (13). 90%. 1H NMR (300 MHz, CD3OD) d: 7.59 (s, 1H),
6.84 (s, 1H), 6.00 (s, 2H), 5.87 (s, 1H), 4.80 (d, J = 15.6 Hz, 1H), 4.60
(d, J = 13.6 Hz, 1H), 4.50 (d, J = 12.4 Hz, 1H), 4.45 (s, 1H), 4.34 (d,
J = 15.8 Hz, 1H), 4.18 (s, 1H), 3.21 (d, J = 12.9 Hz, 1H). 13C NMR
(100 MHz, CD3OD) d: 163.2, 151.1, 146.6, 139.4, 135.0, 125.2,
121.4, 107.6, 104.1, 72.4, 69.8, 68.8, 59.7, 52.0, 42.8. HRMS m/z
(ESI+) calcd for C16H15NO6 (M+Na+) 340.0797, found 340.0807.
4.1.1. (1S,2S,3a1S,12bS)-2,3a1,4,5,7,12b-Hexahydro-1H-[1,3]diox-
olo[4,5-j]pyrrolo[3,2,1-de]phenanthridine-1,2-diyl diacetate (2)
This compound was prepared using a published procedure.24
4.1.2. Dihydroxylation of the C3–C3a olefin
Diacetyl lycorine (2) (0.096 g, 0.258 mmol) was dissolved in
acetone (6 mL) and to this solution of OsO4 (0.25 mL, 2.5% wt solu-
tion in t-BuOH), 0.038 g of NMO (0.284 mmol) of water (1.5 mL)
were added over 5 min. The solution was stirred at room temper-
ature for 14 h, and then the volatiles were evaporated under re-
duced pressure. To the obtained slurry were added EtOAc
(20 mL) and saturated NH4Cl (5 mL), followed by washing with sat-
urated NaCl (10 mL). The extraction was repeated twice with
EtOAc (15 mL) and the combined organic phases were dried with
MgSO4. The residue after evaporation of the solvent was subjected
to silica gel column chromatography (CH2Cl2, then CH2Cl2/
MeOH = 96/4) yielded 3 and 4.
4.1.3.5. (1S,2S,3a1S,12bS)-7-Oxo-2,3a1,4,5,7,12b-hexahydro-1H-
[1,3]dioxolo[4,5-j]pyrrole[3,2,1-de]phenanthridine-1,2-diyl
diacetate (6). This compound was prepared using a published
procedure.10
4.1.4. Preparation of thiolactam 8
Compound 6 (0.0385 g, 0.1 mmol) was suspended in dry ben-
zene (10 mL), then phosphorous pentasulfide (0.02 g, 0.06 mmol)
and hexamethyldisiloxane (0.02 g, 0.16 mmol) was added. The
mixture was refluxed under N2 for 2 h, after that solvent was re-
moved under reduced pressure. The crude residue was purified
by PTLC (CH2Cl2:MeOH = 99/1, Rf = 0.45) to give 0.035 g (88%) of
8 as a yellow solid.
4.1.2.1.
(1S,2S,3S,3aR,3a1S,12bS)-3,3a-Dihydroxy-7-oxo-2,3,3a,
3a1,4,5,7,12b-octahydro-1H-[1,3]dioxolo[4,5-j ]pyrrolo[3,2,1-de]
phenanthridine-1,2-diyl diacetate (3). 53.2% (0.0577 g) as light
yellow viscous oil. 1H and 13C NMR were identical to those pub-
lished in the literature.5b
4.1.4.1. (1S,2S,3a1S,12bS)-7-Thioxo-2,3a1,4,5,7,12b-hexahydro-
1H-[1,3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridine-1,2-diyl
diacetate (8). 1H NMR (300 MHz, CDCl3) d: 8.01 (s, 1H), 6.60 (s,
1H), 5.98 (d, J = 8.0 Hz, 2H), 5.75 (s, 1H), 5.66 (s, 1H), 5.27 (d,
J = 1.4 Hz, 1H), 4.24–3.95 (m, 3H), 3.06 (dd, J = 1.9 Hz, J = 12.1 Hz,
1H), 2.89 (t, J = 8.6 Hz, 2H), 2.08 (s, 3H), 2.03 (s, 3H). 13C NMR
(75 MHz, CDCl3) d: 194.7, 169.9, 151.1, 147.0, 142.8, 131.1, 127.7,
118.9, 116.4, 112.5, 102.7, 74.7, 69.8, 67.2, 56.9, 51.1, 39.5, 28.6,
21.1. HRMS m/z (ESI+) calcd for C20H19NO6S (M+H+) 402.1011,
found 402.1025.
4.1.2.2. 7-Oxo-5,7-dihydro-4H-[1,3]dioxolo[4,5-j]pyrrolo[3,2,1-
de]phenanthridin-2-yl acetate (4). 12.9% as light yellow viscous
oil. 1H and 13C NMR were identical to those published in the
literature.5b
4.1.3. General procedure for acetate hydrolysis to obtain 5, 7, 9,
13
A fresh 0.5 M solution of LiOH in THF/H2O (1/1) was used and its
volume was adjusted to 1.05 equiv of LiOH per acetate residue in
the starting material (4, 6, 3, 12). The acetates were dissolved in
the required volume of LiOH solution and stirred overnight at
18 °C. After that time the two-layered solution became uniform
and 0.05 g of Aberlyst 15(dry) ion-exchange resin (ACROS) were
added and stirred for 1 h. The resin was filtered (glass filter) and
washed on filter with a mixture of MeOH/CH2Cl2 (1/1, 5 mL). Tolu-
ene (15 mL) was added to the obtained mother liquor and evapo-
rated to dryness to obtain pure deacetylated products 5, 7, 9, and
13.
4.1.5. Synthesis of Compound 10
LiAlH4 (0.01 g, 0.26 mmol) was dissolved in 2 mL of THF and
compound 3 (0.011 g, 0.026 mmol) was added to this solution.
The mixture was refluxed for 5 h under argon, after that quenched
with brine (6 mL) and extracted with EtOAc (3 ꢁ 10 mL). The sol-
vent was evaporated under the reduced pressure to afford
0.0056 g (66%) of derivative 10 as white flakes.
4.1.5.1. (1S,2R,3S,3aR,3a1S,12bS)-2,3,3a,3a1,4,5,7,12b-Octahydro-
1H-[1,3]dioxolo[4,5-j-j]pyrrolo[3,2,1-de]phenanthridine-1,2,3,-
3a-tetraol (10). 1H NMR (300 MHz, CD3OD) d: 6.95 (s, 1H), 6.76 (s,
1H), 5.94 (s, 2H), 4.51 (d, J = 13.3 Hz, 1H), 4.21 (d, J = 15.1 Hz, 1H),
4.11 (m, 1H), 3.75 (d, J = 5.2 Hz, 1H), 3.57 (m, 1H), 3.15 (d,
J = 8.7 Hz, 1H), 3.09 (d, J = 8.7 Hz, 1H). 13C NMR (100 MHz, CD3OD)
d: 151.9, 150.9, 148.4, 126.6, 107.4, 105.8, 101.6, 78.6, 69.7, 69.0,
61.4, 54.5, 52.5, 39.1, 35.3, 28.8. HRMS m/z (ESI+) calcd for
4.1.3.1. 2-Hydroxy-4H-[1,3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phe-
nanthridin-7(5H)-one (5). 93%. 1H and 13C NMR were identical
to those published in the literature.25
4.1.3.2. (1S,2S,3a1S,12bS)-1,2-Dihydroxy-3a1,4,5,12b-tetrahydro-
1H-[1,3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridin-7(2H)-
one (7). 95%. 1H and 13C NMR were identical to those published in
the literature.5b
C
16H19NO6 (M+H+) 322.1281, found 322.1291.
4.1.5.2. (1S,2R,3S,3aR,3a1S,12bS)-3a-Hydroxy-7-oxo-2,3,3a,3a1,4,
5,7,12b-octahydro-1H -[1,3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phe-
nanthridine-1,2,3-triyl triacetate (11). This compound was pre-
pared using a published procedure.9
4.1.3.3.
(1S,2R,3S,3aR,3a1S,12bS)-1,2,3,3a-Tetrahydroxy-
3,3a,3a1,4,5,12b-hexahydro-1H-[1,3]dioxolo[4,5-j]pyrrolo[3,2,1-
de]phenanthridin-7(2H)-one (9). 93%. 1H NMR (300 MHz, CD3OD)
d: 7.31 (s, 1H), 6.89 (s, 1H), 6.01 (s, 2H), 4.39 (d, J = 3.0 Hz, 1H), 4.08
(dd, J = 7.4 Hz, 1H), 3.90 (dd, J = 9.1 Hz, 2H), 3.62 (d, J = 9.4 Hz, 1H),
3.48 (quintet, J = 5.8 Hz, 1H), 3.08 (d, J = 14.0 Hz, 1H), 2.14 (quintet,
J = 6.0 Hz, 2H). 13C NMR (75 MHz, CD3OD) d: 162.5, 151.0, 146.6,
134.1, 124.1, 107.1, 105.9, 101.8, 79.2, 77.0, 75.1, 71.4, 63.8, 43.6,
38.5, 22.8. HRMS m/z (ESI+) calcd for C16H17NO7 (M+Na+)
358.0903, found 358.0905.
4.1.5.3. (1S,2R,3R,3a1S,12bS)-7-Oxo-2,3,3a1,5,7,12b-hexahydro-
1H-[1,3]dioxolo[4,5-j-j]pyrrole[3,2,1-de]phenanthridine-1,2,3-
triyl triacetate (12). This compound was prepared using a pub-
lished procedure.9
4.1.6. Synthesis of Compound 14
An oven-dry flask was charged with lycorine (1) (0.0143 g,
0.049 mmol) and Dess–Martin periodinane (0.044 g, 0.103 mmol)

7258
N. M. Evdokimov et al. / Bioorg. Med. Chem. 19 (2011) 7252–7261
and then purged with argon. Dry toluene (2 mL) was then added
and the reaction suspension was heated for 2 h (complete con-
sumption of 1 by TLC). The mixture was cooled to rt and diluted
with EtOAc (10 mL), followed by washing with saturated NaHCO3
(15 mL) and brine, drying over MgSO4, and concentration under re-
duced pressure. The residue was purified on silica gel column with
CH2Cl2:MeOH = 9/1 as an eluent to isolate 0.0093 g (66%) of 14
(Rf = 0.63).
J = 9.0 Hz, J = 17.0 Hz, 1H), 1.09 (s, 21H). 13C NMR (75 MHz, CDCl3)
d: 146.7, 146.3, 141.8, 130.5, 128.0, 118.5, 107.9, 104.5, 101.1, 72.6,
72.3, 61.1, 57.3, 54.0, 41.1, 28.7, 18.2. HRMS m/z (ESI+) calc’d for
C
25H37NO4Si (M+H+) 444.2570, found 444.2576.
4.1.9. Synthesis of Compound 17
To a suspension of lycorine (1) (0.0287 g, 0.1 mmol) and DMAP
(0.0005 g) in dry pyridine (1.5 mL) was added benzoyl chloride
(0.038 mL, 0.33 mmol) at rt. Reaction was stirred at 40 °C for 5 h,
after that time it was diluted with toluene (20 mL) and evaporated
on the rotary evaporator. The residue was dissolved in CH2Cl2
(20 mL) and washed with brine (10 mL) and dried over MgSO4.
The solution was concentrated and purified by column chromatog-
raphy (CH2Cl2) yielding 0.04 g (83%) of compound 17.
4.1.6.1. (1S,3a1S,12bS)-1-Hydroxy-4,5,7,12b-tetrahydro-1H-[1,3]
dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridin-2(3a1H)-one
(14). Pink amorphous solid. 1H and 13C NMR were identical to
those published in the literature.7a
4.1.7. Discrimination of the C1 and C2-hydroxyls, synthesis of
compounds 15 and 19
4.1.9.1.
(1S,2S,3a1S,12bS)-2,3a1,4,5,7,12b-Hexahydro-1H-[1,3]-
Lycorine (1) (0.014 g, 0.049 mmol) was mixed with pyridine
(0.5 mL) and DMAP (0.0001 g). To the obtained mixture pivaloyl
chloride (0.0066 mL, 0.054 mmol) or benzoyl chloride (0.0063
mL, 0.054 mmol) was added in one portion at rt and then stirred
for 12 h under the argon. The residue obtained after rotary evapo-
ration was separated by PTLC to afford 15 (CH2Cl2:MeOH = 49/1;
Rf = 0.49) or 19 (CH2Cl2:MeOH = 97/3; Rf = 0.40).
dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridine-1,2-diyl diben-
zoate (17). White amorphous material. 1H NMR (300 MHz, CDCl3)
d: 8.08 (d, J = 8.0 Hz, 2H), 7.91 (d, J = 8.0 Hz, 2H), 7.56–7.37 (m,
6H), 6.86 (s, 1H), 6.58 (s, 1H), 6.15 (s, 1H), 5.87 (s, 1H), 5.84 (s,
1H), 5.69 (s, 2H), 4.24 (d, J = 14.0 Hz, 1H), 3.67 (d, J = 14.1 Hz,
1H), 3.46 (s, 1H), 3.18 (d, J = 9.9 Hz, 1H), 3.12 (d, J = 9.8 Hz, 1H),
2.75 (s, 2H), 2.58 (s, 1H). 13C NMR (75 MHz, CDCl3) d: 165.5,
165.3, 146.8, 146.5, 146.1, 133.3, 129.8, 128.4, 126.4, 113.9,
107.4, 105.1, 101.2, 70.9, 69.4, 61.7, 56.9, 53.5, 40.8, 28.9.
HRMS m/z (ESI+) calcd for C30H25NO6 (M+H+) 496.1760, found
496.1775.
4.1.7.1. (1S,2S,3a1S,12bS)-1-Hydroxy-2,3a1,4,5,7,12b-hexahydro-
1H-[1,3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridin-2-yl piva-
late (15). 0.013 g (73%) as a yellow oil. 1H NMR (300 MHz, CDCl3)
d: 6.79 (s, 1H), 6.60 (s, 1H), 5.93 (d, J = 6.3 Hz, 2H), 5.46 (s, 1H),
5.43 (s, 1H), 4.45 (s, 1H), 4.14 (d, J = 14.3 Hz, 1H), 3.56 (d,
J = 14.3 Hz, 1H), 3.38 (q, 1H), 2.81 (d, J = 9.1 Hz, 1H), 2.69 (m, 3H),
1.19 (s, 9H). 13C NMR (100 MHz, CDCl3) d: 178.3, 146.6, 146.4,
145.8, 129.9, 127.1, 113.7, 107.8, 104.5, 101.1, 73.2, 69.4, 60.7,
57.7, 53.7, 41.0, 39.0, 29.0, 27.9. HRMS m/z (ESI+) calcd for
4.1.10. Synthesis of Compound 18
To a solution of compound 17 (0.03 g, 0.06 mmol) in MeOH
(3 mL) was added LiOH monohydrate (0.0025 g, 0.06 mmol) at rt.
After 3 h the reaction mixture was subjected to PTLC (CH2Cl2:
MeOH = 48/3) to isolate compound 18 (33%, Rf = 0.71) and com-
pound 19 (56%, Rf = 0.56).
C
21H25NO5 (M+H+) 372.1811, found 372.1812.
4.1.7.2. (1S,2S,3a1S,12bSbS)-1-hydroxy-2,3a1,4,5,7,12b-hexahy-
dro-1H-[1,3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridin-2-yl
benzoate (19).
4.1.10.1.
(1S,2S,3a1S,12bS)-2-Hydroxy-2,3a1,4,5,7,12b-hexahy-
0.0136 g (68%) as a yellow oil. 1H NMR
dro-1H-[1,3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridin-1-yl
benzoate (18). 0.007 g (33%) white amorphous solid. 1H and 13C
NMR were identical to those published in the literature.5a
(300 MHz, CDCl3) d: 8.05 (d, J = 7.4 Hz, 2H), 7.54 (q, J = 7.4 Hz,
1H), 7.43 (t, J = 7.7 Hz, J = 15.1 Hz, 2H), 6.84 (s, 1H), 6.63 (s, 1H),
5.92 (s, 2H), 5.65 (s, 1H), 5.59 (s, 1H), 4.70 (s, 1H), 4.21 (t,
J = 3.6 Hz, J = 12.4 Hz, 1H), 3.66 (d, J = 3.4 Hz, 1H), 2.76 (s, 1H),
2.30 (d, J = 7.4 Hz, 1H), 2.02 (s, 1H), 1.40 (d, J = 7.0 Hz, 2H), 1.21
(d, J = 7.4 Hz, 1H). 13C NMR (100 MHz, CDCl3) d: 166.1, 149.2,
147.5, 147.3, 133.8, 129.7, 128.8, 128.5, 118.0, 108.8, 105.8,
101.2, 73.3, 68.2, 53.6, 53.3, 40.5, 29.7. HRMS m/z (ESI+) calc’d for
4.1.11. General procedure for acylation of compound 16.
Synthesis of compounds 20 and 21
Compound 16 (0.032 g, 0.072 mmol) was co-evaporated with
toluene (5 mL) and dissolved in CH2Cl2 (10 mL). Selected carboxylic
acid (0.08 mmol), DCC (0.018 g, 0.085 mmol) and DMAP (0.0005 g)
were added at 0 °C. The mixture was kept under argon at rt for
12 h. In the case of compounds 20 and 21 the mixture was sub-
jected to column chromatography using CHCl3 as a solvent to ob-
tain pure 20 and 21. In the other cases, the crude material was
used in the next step.
C
23H21NO5 (M+H+) 392.1498, found 392.1486.
4.1.8. Synthesis of Compound 16
To a solution of lycorine (1) (0.0287 g, 0.1 mmol) in DMF (0.22
mL) under argon, pyridine (0.025 mL, 0.31 mmol), AgNO3
(0.0685 g, 0.4 mmol) and tri-iso-propysilyl chloride (0.042 mL,
0.2 mmol) were added at 20 °C and the reaction was kept at this
temperature in a dark place for 4 h. Then saturated NH4Cl solution
(5 mL) was added and organic phase was extracted with Et2O
(3 ꢁ 10), washed with brine (10 mL). The ether was then removed
under reduced pressure and crude was separated by flash column
chromatography (CHCl3:MeOH:NH4OH = 95/5/0.5) to give 0.0351 g
(79%) of compound 16 (Rf = 0.48).
4.1.11.1.
(1S,2S,3a1S,12bS)-2-(Triisopropylsilyloxy)-2,3a1,4,5,7,
12b-hexahydro-1H-[1,3]dioxolo [4,5-j]pyrrolo[3,2,1-de]phenan-
thridin-1-yl palmitate (20). 0.035 g (71%) as a colorless oil. 1H
NMR (300 MHz, CDCl3) d: 6.75 (s, 1H), 6.55 (s, 1H), 5.90 (s, 2H),
5.69 (s, 1H), 5.50 (s, 1H), 4.30 (s, 1H), 4.12 (d, J = 14.3 Hz, 1H),
3.52 (d, J = 14.1 Hz, 1H), 3.36 (m, 1H), 2.97 (d, J = 10.4 Hz, 1H),
2.76 (d, J = 10.5 Hz, 1H), 2.63 (m, 2H), 2.28 (t, J = 8.9 Hz,
J = 16.8 Hz, 1H), 2.17 (t, J = 5.8 Hz, J = 14.6 Hz, 2H), 1.60 (q, 2H),
1.44 (q, J = 7.7 Hz, 2H), 1.25 (s, 21H), 1.08 (d, J = 5.5 Hz, 24H),
0.87 (t, J = 6.6 Hz, J = 13.2 Hz, 3H). 13C NMR (75 MHz, CDCl3) d:
173.2, 146.5, 146.2, 142.2, 129.5, 127.8, 118.4, 107.3, 105.1,
101.0, 72.0, 69.9, 61.9, 57.0, 53.9, 39.5, 34.5, 32.0, 29.8, 29.5, 28.9,
28.7, 25.1, 22.8, 18.2, 14.2. HRMS m/z (ESI+) calcd for C41H67NO5Si
(M+H+) 682.4867, found 682.4873.
4.1.8.1.
(1S,2S,3a1S,12bS)-2-(Triisopropylsilyloxy)-2,3a1,4,5,7,
12b-hexahydro-1H-[1,3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenan-
1
thridin-1-ol (16). H NMR (300 MHz, CDCl3) d: 6.81 (s, 1H), 6.59 (s,
1H), 5.92 (s, 2H), 5.50 (s, 1H), 4.50 (s, 1H), 4.39 (s, 1H), 4.16 (d,
J = 13.8 Hz, 1H), 3.51 (d, J = 13.6 Hz, 1H), 3.34 (m, 1H), 2.86 (d,
J = 12.9 Hz, 1H), 2.73 (d, J = 12.7 Hz, 1H), 2.61 (m, 2H), 2.35 (t,

N. M. Evdokimov et al. / Bioorg. Med. Chem. 19 (2011) 7252–7261
7259
4.1.11.2. (1S,2S,3a1S,12bS)-2-(Triisopropylsilyloxy)-2,3a1,4,5,7,
12b-hexahydro-1H-[1,3]dioxolo [4,5-j]pyrrolo[3,2,1-de]phenan-
thridin-1-yl acetate (21).
12H), 4.18 (s, 1H), 4.11 (d, J = 14.0 Hz, 1H), 3.52 (d, J = 14.0 Hz,
1H), 3.36 (q, J = 4.1 Hz, 1H), 2.84–2.62 (m, 16H), 2.24 (m, 3H),
2.74 (t, J = 7.4 Hz, 1H), 0.96 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz,
CDCl3) d: 172.8, 146.6, 146.3, 143.8, 129.3, 128.7, 128.3, 128.2,
127.1, 117.5, 107.4, 105.1, 101.0, 72.7, 69.6, 61.7, 56.9, 53.8, 39.4,
34.2, 28.7, 25.7, 25.6, 22.8, 20.6, 14.4. HRMS m/z (ESI+) calc’d for
0.028 g (80%) as an oil. 1H NMR
(300 MHz, CDCl3) d: 6.75 (s, 1H), 6.56 (s, 1H), 5.91 (d, J = 1.4 Hz,
2H), 5.66 (s, 1H), 5.50 (s, 1H), 4.32 (s, 1H), 4.17 (d, J = 14.0 Hz,
1H), 3.54 (d, J = 14.0 Hz, 1H), 3.36 (m, 2H), 3.01 (d, J = 10.2 Hz,
1H), 2.77 (d, J = 10.2 Hz, 1H), 2.63 (m, 2H), 2.39 (t, J = 9.1 Hz, 1H),
1.93 (s, 3H), 1.08 (m, 21H). 13C NMR (75 MHz, CDCl3) d: 170.5,
146.2, 142.3, 129.5, 127.8, 118.2, 107.4, 105.0, 100.7, 72.4, 69.8,
61.7, 57.2, 53.9, 39.4, 28.7, 21.2, 18.2. HRMS m/z (ESI+) calcd for
C
38H47NO5 (M+H+) 598.3532, found 598.3538.
4.1.13. Synthesis of compound 27
Compound 16 (0.032 g, 0.072 mmol) was dissolved in dry DMF
(0.5 mL). To the obtained solution under argon at 20 °C were added
NaH (0.008 g, 0.2 mmol, 60% suspension in mineral oil) and after
10 min benzyl bromide (0.012 mL, 0.1 mmol). The reaction was left
stirring for 20 h at rt. The mixture was diluted with 20 ml of 2-pro-
panol and solvents were removed at reduced pressure. Remaining
residue was dissolved in THF (3 mL) and TBAF (0.066 mL,
0.066 mmol, 1.0 M solution in THF) was added to the obtained
solution dropwise at rt. The reaction was left for 4 h at this temper-
ature, then quenched with saturated solution of NH4Cl (5 mL) and
extracted with EtOAc (3 ꢁ 5 mL). Column chromatography
(CH2Cl2:MeOH = 48/2) afforded pure 27.
C
27H39NO5Si (M+H+) 486.2676, found 486.2664.
4.1.12. General procedure for the removal of the TIPS-protecting
group. Synthesis of compounds 22, 23, 24, 25, 26
Pure TIPS-protected 20, 21 or crude mixtures for the other
C1-acylated lycorines from the procedure above (0.06 mmol) were
dissolved in THF (3 mL) and TBAF (0.066 mL, 0.066 mmol, 1.0 M
solution in THF) was added to the obtained solution dropwise at
rt. The reaction was left for 4 h at this temperature, then quenched
with saturated solution of NH4Cl (5 mL) and extracted with EtOAc
(3 ꢁ 5 mL). Column chromatography (CH2Cl2:MeOH = 48/2) affor-
ded pure C1-acylated lycorines 22, 23, 24, 25, 26.
4.1.13.1. (1S,2S,3a1S,12bS)-1-(Benzyloxy)-2,3a1,4,5,7,12b-hexa-
hydro-1H-[1,3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridin-2-
4.1.12.1. (1S,2S,3a1S,12bS)-2-Hydroxy-2,3a1,4,5,7,12b-hexahy-
dro-1H-[1,3]dioxolo[4,5-j-j]pyrrole [3,2,1-de]phenanthridin-1-
ol (27).
38% as a yellow oil. 1H NMR (300 MHz, CDCl3) d:
yl palmitate (22).
88% as a yellow oil. 1H and 13C NMR were
7.28–7.25 (m, 5H), 6.57 (s, 1H), 6.49 (s, 1H), 5.92 (s, 2H), 5.63 (s,
1H), 4.66 (d, J = 11.8 Hz, 1H), 4.42 (s, 1H), 4.27 (s, 1H), 4.13 (d,
J = 14.3 Hz, 1H), 3.41 (m, 1H), 4.63 (d, J = 13.9 Hz, 1H), 2.67 (s,
2H). 13C NMR (100 MHz, CDCl3) d: 149.8, 146.0, 144.4, 137.9,
135.4, 128.3, 128.2, 128.1, 128.0, 127.7, 125.6, 117.9, 107.2,
105.3, 101.0, 72.2, 72.0, 67.9, 53.5, 52.6, 50.4, 29.4. HRMS m/z
(ESI+) calcd for C23H23NO4 (M+H+) 378.1705, found 378.1713.
identical to those published in the literature.26
4.1.12.2. (1S,2S,3a1S,12bS)-2-Hydroxy-2,3a1,4,5,7,12b-hexahy-
dro-1H-[1,3]dioxolo[4,5-j]pyrrole [3,2,1-de]phenanthridin-1-yl
acetate (23).
81% as a yellow oil. 1H and 13C NMR were iden-
tical to those published in the literature.5b
4.1.12.3. (1S,2S,3a1S,12bS)-2-Hydroxy-2,3a1,4,5,7,12b-hexahy-
dro-1H-[1,3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridin-1-yl
stearate (24).
4.1.14. Synthesis of compound 28
To a solution of compound 7 (0.0141 g, 0.0468 mmol) in 0.5 mL
of dry pyridine at 20 °C was added thionyl chloride (0.1 mL). After
4 h of stirring at 20 °C the mixture was separated by PTLC
(CH2Cl2:MeOH = 49/1) give 0.011 g (92%) of compound 28
(Rf = 0.51) as a white solid.
67% as a viscous oil. 1H NMR (300 MHz, CDCl3)
d: 6.69 (s, 1H), 6.56 (s, 1H), 5.91 (d, J = 2.5 Hz, 2H), 5.65 (s, 1H), 5.55
(s, 1H), 4.21 (s, 1H), 4.17 (d, J = 14 Hz, 1H), 3.54 (d, J = 14 Hz, 1H),
3.36 (q, 1H), 2.89 (d, J = 10.4 Hz, 1H), 2.75 (d, J = 10.4 Hz, 1H),
2.64 (m, 2H), 2.41 (t, J = 6.8 Hz, 1H), 2.18 (t, J = 7.4 Hz, 1H), 1.43
(t, J = 6.6 Hz, 2H), 1.25 (s, 32H), 1.14 (s, 2H), 0.88 (t, J = 6.6 Hz,
3H). 13C NMR (75 MHz, CDCl3) d: 173.6, 146.6, 146.3, 141.8,
129.2, 127.2, 117.2, 107.4, 105.1, 101.0, 72.4, 70.0, 61.7, 57.0,
53.8, 39.6, 34.4, 32.0, 29.8, 29.5, 29.3, 28.8, 25.1, 22.8, 14.2. HRMS
m/z (ESI+) calcd for C34H51NO5 (M+H+) 554.3845, found 554.3854.
4.1.14.1. 4H-[1,3]Dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridin-
7(5H)-one (28). 92%; 1H and 13C NMR were identical to those pub-
lished in the literature.12
4.1.15. Synthesis of compounds 29 and 30
Selected compound 28 (0.0048 g, 0.018 mmol) or 4 (0.0058 g,
0.018 mmol) was refluxed under N2 in dry benzene (1 mL) with
DDQ (0.126 mmol) for 24 h. After that time the reaction
mixtures were diluted with EtOAc (7 mL) and washed with
saturated aqueous solution of Na2SO3 (6 mL), then saturated
solution of NaHCO3 (6 mL) followed by brine (3 mL). The organic
phase was concentrated in vacuum and subjected to PTLC
(CH2Cl2:MeOH = 99/1) to afford pure compounds 29 and 30
respectively.
4.1.12.4. (1S,2S,3a1S,12bS)-2-Hydroxy-2,3a1,4,5,7,12b-hexahy-
dro-1H-[1,3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridin-1-yl
oleate (25).
52% as a colorless oil. 1H NMR (300 MHz, CDCl3)
d: 6.68 (s, 1H), 6.56 (s, 1H), 5.90 (s, 2H), 5.64 (s, 1H), 5.55 (s, 1H),
5.34 (d, J = 3.84 Hz, 2H), 4.19 (s, 1H), 4.12 (d, J = 14.5 Hz, 1H),
3.53 (d, J = 14.5 Hz, 1H), 3.34 (q, J = 9.1 Hz, 1H), 2.85 (d, J = 9.6 Hz,
1H), 2.76 (d, J = 9.6 Hz, 1H), 2.63 (m, 2H), 2.40 (t, J = 8.8 Hz, 1H),
2.17 (t, J = 7.1 Hz, J = 14.5 Hz, 2H), 1.99 (m, 4H), 1.45 (t, J = 7.1 Hz,
J = 13.5 Hz, 2H), 1.26 (s, 20H), 1,15 (s, 2H), 0.87 (t, J = 5.5 Hz,
J = 12.1 Hz, 3H). 13C NMR (75 MHz, CDCl3) d: 173.6, 146.5, 144.5,
130.1, 129.8, 129.5, 127.2, 117.2, 107.3, 105.1, 101.0, 72.4, 69.9,
61.7, 57.0, 53.8, 39.6, 34.4, 32.0, 29.9, 29.6, 29.4, 29.2, 27.3, 25.0,
22.8, 14.2. HRMS m/z (ESI+) calcd for C34H49NO5 (M+H+)
552.3689, found 552.3691.
4.1.15.1. 7H-[1,3]Dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridin-
7-one (29). 0.0039 g (82%) as a white solid. 1H and 13C NMR were
identical to those published in the literature.12
4.1.15.2. 7-Oxo-7H-[1,3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenan-
thridin-2-yl acetate (30). 0.0054 g (95%) as a white solid. 1H
NMR (300 MHz, CDCl3) d: 8.06 (d, J = 3.6 Hz, 1H), 7.97 (s, 1H),
7.62 (d, J = 1.7 Hz, 1H), 7.55 (s, 1H), 7.46 (d, J = 1.7 Hz, 1H),
6.87 (d, J = 3.6 Hz, 1H), 6.17 (s, 2H), 2.39 (s, 3H). 13C NMR
(75 MHz, CDCl3) d: 194.4, 170.0, 152.9, 148.8, 147.4, 131.0,
128.9, 124.7, 122.8, 122.6, 115.9, 112.3, 110.8, 108.3, 102.5,
4.1.12.5. (4Z,7Z,10Z,13Z,16Z,19Z)-((1S,2S,3a1S,12bS)-2-Hydroxy-
2,3a1,4,5,7,12b-hexahydro-1H-[1,3]dioxolo[4,5-j]pyrrolo[3,2,1-
de]phenanthridin-1-yl)
(26).
65% as an oil. 1H NMR (300 MHz, CDCl3) d: 6.66 (s,
1H), 6.55 (s, 1H), 5.90 (s, 2H), 5.63 (s, 1H), 5.53 (s, 1H), 5.37 (m,
docosa-4,7,10,13,16,19-hexaenoate

7260
N. M. Evdokimov et al. / Bioorg. Med. Chem. 19 (2011) 7252–7261
102.1, 21.3. HRMS m/z (ESI+) calcd for C18H11NO5 (M+H+)
322.0715, found 322.0726.
1H), 5.91 (s, 2H), 5.89 (s, 1H), 5.75 (dd, J = 4.1 Hz, J = 11.1 Hz, 1H),
4.13 (q, J = 7.1 Hz, J = 14.3 Hz, 2H), 4.05 (d, J = 14.5 Hz, 1H), 3.44
(m, 2H), 3.18 (dd, J = 7.1 Hz, 1H), 2.70 (d, J = 14.6 Hz, 1H), 2.61 (s,
1H), 2.57 (m, 2H), 2.28 (m, 2H), 1.82 (s, 3H), 1.24 (t, J = 7.1 Hz,
J = 14.3 Hz, 3H). 13C NMR (75 MHz, CDCl3) d: 172.5, 170.7, 146.9,
146.1, 138.6, 136.3, 132.3, 125.8, 107.2, 106.7, 101.0, 67.4, 67.2,
60.4, 57.2, 53.3, 43.0, 40.0, 37.5, 31.7, 21.1, 14.3. HRMS m/z (ESI+)
calcd for C22H25NO6 (M+H+) 400.1760, found 400.1769.
4.1.16. Synthesis of compound 31
n-Butyllithium (0.09 mL, 0.22 mmol, 2.5 M solution in hexanes)
was added under argon to a cold (0 °C) suspension of lycorine (1)
(0.0287 g, 0.1 mmol) in THF (3 mL). The resulting solution was stir-
red at 0 °C for 20 min and then transferred to a solution of BOC
anhydride (0.048 g, 0.21 mmol) in THF (5 mL) at 20 °C, then the
mixture was stirred at this temperature for 5 h. The reaction was
quenched with brine (2 mL), and extracted with EtOAc followed
by washing with brine (3 ꢁ 10 mL), then dried (MgSO4). Rotary
evaporation of the volatiles provided 0.048 g (98%) of compound
31 as a light yellow oil.
4.2. Determination of GI50 growth inhibitory values
The overall growth levels of each human cancer cell line
was determined using the colorimetric MTT (3-[4,5-dimethyl-
thiazol-2yl])-diphenyl tetrazolium bromide, Sigma, Belgium)
assay.6h,8,13a,17 Briefly, the cell lines were incubated for 24 h in
96-microwell plates (at a concentration of 10,000–40,000 cells
per 1 mL of culture medium depending on the cell type) to ensure
adequate plating prior to cell growth determination. The assess-
ment of cell population growth by means of the MTT assay is based
on the capability of living cells to reduce the yellow MTT reagent to
the blue product, formazan, by a reduction reaction occurring in
the mitochondria. The number of living cells after 72 h of culture
in the presence (or absence: control) of the various compounds is
directly proportional to the intensity of the blue staining, which
is quantitatively measured by spectrophotometry—in our case
using a Biorad Model 680XR (Biorad, Nazareth, Belgium)—at a
570 nm wavelength (with a reference of 630 nm). Each set of
experimental conditions was evaluated in sextuplicate.6h,8,13a,17
4.1.16.1. tert-Butyl (1S,2S,3a1S,12bSbS)-2,3a1,4,5,7,12b-hexahy-
dro-1H-[1,3]dioxolo[4,5-j]pyrrole[3, 2,1-de]phenanthridine-1,2-
diyl dicarbonate (31). 1H NMR (300 MHz, CDCl3) v: 6.78 (s, 1H),
6.55 (s, 1H), 5.89 (d, J = 1.92 Hz, 2H), 5.60 (s, 1H), 5.57 (s, 1H),
5.15 (s, 1H), 4.09 (d, J = 13.5 Hz, 1H), 3.54 (d, J = 13.5 Hz, 1H),
3.32 (m, 1H), 2.87 (d, J = 10.4 Hz, 1H), 2.80 (d, J = 10.5 Hz, 1H),
2.61 (m, 2H), 2.38 (t, J = 8.52 Hz, 1H), 1.50 (s, 9H), 1.41 (s, 9H).
13C NMR (75 MHz, CDCl3) v: 152.9, 152.6, 146.7, 146.4, 129.7,
126.7, 113.5, 107.4, 105.3, 101.0, 82.9, 82.8, 73.6, 72.5, 61.1, 57.0,
53.7, 40.6, 28.9, 27.9. HRMS m/z (ESI+) calcd for C26H33NO8
(M+H+) 488.2284, found 488.2294.
4.1.17. Synthesis of compound 32
To a solution of lycorine (1) (0.0287 g, 0.1 mmol) in 0.5 mL of dry
DMF under argon were added at 20 °C NaH (0.016 g, 0.4 mmol, 60%
suspension in mineral oil) and after 10 min allyl bromide (0.02 mL,
0.2 mmol). The reaction was left stirring for 20 h at rt. The mixture
was diluted with 20 mL of 2-propanol and the solvents were
removed at reduced pressure. PTLC (CH2Cl2: MeOH = 48/2, Rf =
0.25) afforded 0.013 g (35%) of compound 32 as a yellow oil.
Acknowledgments
This work is supported by the US National Institutes of Health
(RR-16480 and CA-135579) under the BRIN/INBRE and AREA pro-
grams as well as by the Fonds Yvonne Boël (Brussels, Belgium).
V. M. and R. K. are a post-doctoral fellow and the director of re-
search with the Fonds National de la Recherche Scientifique (FNRS,
Belgium). In addition, grants from the Italian Ministry of University
and Research are gratefully acknowledged.
4.1.17.1.
(1S,2S,3a1R,12bS)-1,2-bis(allyloxy)-2,3a1,4,5,7,12b-
hexahydro-1H-[1,3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthri-
dine (32). 1H NMR (300 MHz, CDCl3) d: 6.78 (s, 1H), 6.57 (s, 1H),
5.98–5.75 (m, 4H), 5.56 (s, 1H), 5.35–5.17 (m, 4H), 4.23–4.03 (m,
7H), 3.56 (d, J = 11.6 Hz, 1H), 3.31 (dd, J = 6.6 Hz, 1H), 2.82 (d,
J = 9.6 Hz, 1H), 2.75 (t, J = 9.9 Hz, 1H), 2.62 (m, 3H), 2.40 (s, 1H).
13C NMR (100 MHz, CDCl3) d: 146.2, 145.9, 135.1, 135.0, 128.6,
117.7, 117.1, 115.6, 105.1, 100.8, 75.4, 75.1, 71.1, 70.5, 61.4, 53.7,
41.0, 28.7. HRMS m/z (ESI+) calcd for C22H25NO4 (M+H+)
368.1862, found 368.1869.
A. Supplementary data
Supplementary data (copies of 1H and 13C NMR data for all the
new compounds) associated with this article can be found, in the
online version, at doi:10.1016/j.bmc.2011.09.051.
References and notes
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