Stereospecific synthesis and biological evaluation of monodesmethyl metabolites of (+)-13a-(S)-deoxytylophorinine as potential antitumor agents

Article abstract of DOI:10.1055/s-0032-1316810

Three major monodesmethyl metabolites of (+)-13a-(S)-deoxytylophorinine were synthesized stereospecifically and their configurations at C-13a were determined. Biological assays revealed that one of the metabolites, 3-O-desmethyl-13a-(S)-deoxytylophorinine

Full text of DOI:10.1055/s-0032-1316810

PAPER
▌3757
paper
Stereospecific Synthesis and Biological Evaluation of Monodesmethyl Metabo-
lites of (+)-13a-(S)-Deoxytylophorinine as Potential Antitumor Agents
Monodesmethyl Metabolites of (+)-13a-(S)-Deoxytylophorinine
Pengfei Yu,1 Haining Lv,1 Chao Li, Jinhong Ren, Shuanggang Ma, Song Xu, Xiaoguang Chen, Shishan Yu*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical
Sciences and Peking Union Medical College, Beijing 100050, P. R. of China
Fax +86(10)63017757; E-mail: yushishan@imm.ac.cn
Received: 09.09.2012; Accepted after revision: 24.10.2012
possessed potent cytotoxic activities through blockage of
Abstract: Three major monodesmethyl metabolites of (+)-13a-(S)-
the PI3K and MAPK signaling transduction pathways and
deoxytylophorinine were synthesized stereospecifically and their
by interference with progression of the cell cycle. These
findings identified (S)-1 and its derivatives as promising
configurations at C-13a were determined. Biological assays re-
vealed that one of the metabolites, 3-O-desmethyl-13a-(S)-deoxyty-
lophorinine, had a higher cytotoxic potency than the parent antitumor chemotherapeutic agents and prompted us to in-
compound or the positive controls doxorubicin (Adriamycin) and
paclitaxel (Taxol).
vestigate them further.
Key words: alkaloids, medicinal chemistry, antitumor agents, ste-
MeO
reoselective synthesis
H
N
Historically, natural products and their derivatives have
MeO
served as major sources of therapeutic agents and lead
OMe
molecules in drug discovery. In the early nineteenth cen-
tury, about 80% of all drugs were obtained from roots,
Figure 1 Chemical structure of (S)-1
bark, or leaves. More recently, over 70% of antitumor
To demonstrate the potential of (S)-1 as a candidate for
cancer treatment clinical trials, the absorption, distribu-
tion, metabolism, and excretion properties of this com-
pound were investigated. The compound is susceptible to
metabolism by rat liver microsomes, and the metabolite
mixture is as potent as the parent compound (data not
shown). Compared with the parent compound, some me-
tabolites of (S)-1 might have similar or greater levels of
cytotoxic activity.
compounds are either natural products or derivatives
thereof.2–4 For example, the phenanthroindolizidine alka-
loids are pentacyclic natural products found primarily in
the plant families Cynanchum, Pergularia, and Tylophora
and in some genera of the Asclepiadaceae.5–7 These alka-
loids are known for their extensive bioactivities, especial-
ly their profound antitumor activities.8–12 However, side
effects have limited their application as antitumor drugs.
For example, in the early 1960s, the clinical candidate ty-
locrebrine was found to be toxic to the central nervous
system (CNS), causing disorientation and ataxia.13 As a
result, no phenanthroindolizidine alkaloid is currently
used in clinical treatment. Novel phenanthroindolizidine
alkaloids and their derivatives with potent antitumor ac-
tivities and reduced CNS toxicities are therefore very at-
tractive targets for pharmaceutical research.
Recently, Abliz and co-workers investigated the main
metabolites of (S)-1 in rat urine, and they identified 6-O-
desmethyldeoxytylophorinine (2), 7-O-desmethyldeoxy-
tylophorinine (3), and 3-O-desmethyldeoxytylophorinine
(4) by integrated rapid-resolution liquid chromatography–
tandem mass spectrometry.17 However, the configurations
of these compounds at C-13a and their activities could not
be determined because of the limited quantities that were
available, thereby necessitating the synthesis of these me-
tabolites and their enantiomers (Scheme 1). Furthermore,
with these compounds in hand, structure–activity relation-
ships for (S)-1 and its metabolites could be systematically
studied.
The phenanthroindolizidine alkaloid (+)-13a-(S)-deoxy-
tylophorinine [(S)-1; Figure 1] was originally isolated
from the roots of Tylophora atrofolliculata and T. ovata,
and it was found to possess potent anticancer activities
both in vitro and in vivo.14 Moreover, (S)-1 penetrates the
blood–brain barrier and distributes in brain tissues with-
out obvious CNS toxicity.15 This compound therefore
aroused our particular attention and interest. We recently
reported a synthesis, biological evaluation, and mechanis-
tic study on (S)-1 and its derivatives.16 The biological re-
sults showed that this alkaloid and some of its derivatives
We synthesized 6-O-desmethyldeoxytylophorinine [(S)-2
or (R)-2] and 7-O-desmethyldeoxytylophorinine [(S)-3 or
(R)-3], two of the main metabolites of (S)-1, for the first
time. This synthesis was accomplished by random des-
methylation of (S)-1 or its enantiomer (–)-13a-(R)-deoxy-
tylophorinine [(R)-1] with magnesium iodide under
solvent-free conditions (Scheme 2).18 The demethylated
positions in (S)-2, (R)-2, (S)-3, and (R)-3 were determined
SYNTHESIS 2012, 44, 3757–3764
Advanced online publication: 09.11.2012
0
0
3
9
-
7
8
8
1
1
4
3
7
-
2
1
0
X
DOI: 10.1055/s-0032-1316810; Art ID: SS-2012-H0709-OP
© Georg Thieme Verlag Stuttgart · New York

3758
P. Yu et al.
PAPER
R1O
2
MeO
MeO
1
3
4
H
H
metabolized
in vivo
purification
13a
13a
N
N
metabolites
5
8
6
R2O
7
OR3
OMe
(+)-13a-(S)-deoxytylophorinine (S)-1
potent cytoxicity
2
3
4
R2 = H, R1 = R3 = Me
R3 = H, R1 = R2 = Me
R1 = H, R2 = R3 = Me
Scheme 1 Metabolism of (S)-1. The mixture of metabolites of (S)-1 from rat-liver microsomes showed a comparable cytotoxic potency to that
of the parent compound. After purification, the main metabolites in rat urine were identified by an integrated rapid-resolution liquid chroma-
tography–tandem mass spectrometric approach to be 6-O-desmethyldeoxytylophorinine (2), 7-O-desmethyldeoxytylophorinine (3), and 3-O-
desmethyldeoxytylophorinine (4).
MeO
MeO
After Perkin condensation and esterification, the interme-
diate 9 was subjected to iron(III) chloride catalyzed intra-
molecular oxidative coupling to construct the
phenanthrene ring. Gratifyingly, removal of the isopropyl
group and oxidative coupling were accomplished simulta-
neously. After benzylation of the hydroxy group in the 3-
position and application of analogous steps to those used
in the synthesis of (S)-1,16,21–24 we obtained compound
(S)-5, a novel derivative of (S)-1 with a benzyloxy group
at C-3. The target compound (S)-4 was obtained by cata-
lytic hydrogenation of (S)-5 in 11% overall yield and
>99% ee. Compounds (R)-4 and (R)-5 were prepared by
using the same procedures and materials as (S)-4 and (S)-
5, except for the use of dimethyl D-glutamate hydrochlo-
ride as the building block (ee >99%).
*
*
N
N
MgI2
R1O
MeO
OR2
OMe
(S)-1 >99% ee
(S)-2 R1 = H, R2 = Me, 30%, >99% ee
(S)-3 R1 = Me, R2 = H, 16%, >99% ee
(R)-1 >99% ee
(R)-2 R1 = H, R2 = Me, 26%, >99% ee
(R)-3 R1 = Me, R2 = H, 14%, >99% ee
Scheme 2 Syntheses of (S)-2, (R)-2, (S)-3, and (R)-3
by means of nuclear Overhauser effect (NOE) experi-
ments (see Supporting Information).
Because the C-13a configurations of the metabolites had
not been determined previously17 as a result of the limited
quantities available in rat urine, the purified metabolites
of (S)-1 and the synthesized monodesmethyl compounds
were subjected to chiral-HPLC analysis (CHIRALPAK
AD-H column, 4.6 × 250 mm, 5 µm). By comparing the
retention time of each metabolite with that of the corre-
sponding synthetic enantiomer, the C-3 desmethyl metab-
olite of (S)-1 was found to be identical to (S)-4, and the C-
6 desmethyl metabolite and C-7 desmethyl metabolite
were found to be identical to (S)-2 and (S)-3, respectively.
These results showed that the configuration at C-13a was
unchanged during metabolism.
Circular dichroism (CD) spectra of 1, 2, and 3 were re-
corded (see Supporting Information) to determine wheth-
er the configuration at C-13a was changed during random
demethylation. The CD spectra of (S)-1, (S)-2, and (S)-3
displayed positive Cotton effects in the region 270–280
nm, showing that the absolute configuration at C-13a was
S.19 Moreover, the CD spectra of (R)-2 and (R)-3 dis-
played negative Cotton effects in this region, as did the
parent compound (R)-1. Therefore, the configuration at C-
13a was retained during random demethylation.
Having accomplished stereospecific syntheses of the C-6
and C-7 desmethyl metabolites of (S)-1, we also prepared
3-O-desmethyldeoxytylophorinine [(S)-4 or (R)-4]. Our
synthetic route to this compound was similar to that ad-
opted by Ikeda,19 but with a different protecting group and
different intramolecular oxidative coupling conditions.
The enantiomeric excess (ee) of the final product was
more than 99%. As shown in Scheme 3, the hydroxy
group of 4-hydroxybenzaldehyde (6) was protected with
an isopropyl group to give 4-isopropoxybenzaldehyde (7).
The reason for selecting isopropyl as the protecting group
was that aluminum(III) chloride has been reported to
cleave isopropyl aryl ethers while leaving methyl aryl
ethers intact.20 We surmised that the Lewis acid iron(III)
chloride, an oxidative coupling reagent commonly used in
the synthesis of phenanthrene rings, would function in the
same manner as aluminum(III) chloride in this respect.
Having successfully synthesized the optically pure metab-
olites and derivatives of 13a-(S)-deoxytylophorinine and
their enantiomers, we were interested in investigating the
cytotoxic activities of these compounds [(S)-1–(S)-5 and
(R)-1–(R)-5] with five human cancer cell lines: A375 (hu-
man malignant melanoma cell line), SH-SY5Y (human
neuroblastoma cell line), HepG2 (human hepatocellular
cancer cell line), SKOV3 (human ovarian cancer cell
line), and U251 (human glioblastoma cancer cell line).
This assay was conducted by an in vitro method using
3-(4,5-dimethyl-1,3-thiazol-2-yl)-2,5-diphenyl-2H-tetra-
zol-3-ium bromide> (MTT). Doxorubicin (Adriamycin)
and paclitaxel (Taxol) were used as positive controls. The
concentrations (in nM) that inhibited cell growth by 50%
(IC50) are listed in Table 1.
Synthesis 2012, 44, 3757–3764
© Georg Thieme Verlag Stuttgart · New York

PAPER
Monodesmethyl Metabolites of (+)-13a-(S)-Deoxytylophorinine
3759
O
O
HO
O
O
OH
OMe
OMe
H
H
a
b
c
d
O
O
O
HO
O
MeO
MeO
MeO
OMe
OMe
OMe
6
7 97%
8 69%
9 87%
10
BnO
MeO
BnO
MeO
BnO
MeO
BnO
MeO
OMe
*
OH
*
O
O
OMe
OH
N
N
e
f
g–i
j
O
O
O
OMe
OMe
OMe
OMe
11 52% (2 steps)
12 97%
(S)-13 82% (3 steps)
(R)-13 80% (3 steps)
(S)-14 90%
(R)-14 88%
BnO
MeO
BnO
BnO
HO
O
*
*
*
*
N
N
N
N
k
l, m
n
o
O
O
MeO
MeO
MeO
OMe
OMe
OMe
OMe
(S)-15 84%
(R)-15 82%
(S)-16 93 (2 steps)
(R)-16 91 (2 steps)
(S)-5 84%
(R)-5 81%
(S)-4 76%, >99% ee
(R)-4 75%, >99% ee
Scheme 3 Syntheses of (S)-4, (R)-4, (S)-5, and (R)-5. Reagents and conditions: a) i-PrBr, KI, K2CO3, DMF; b) Ac2O, Et3N; c) (ClCO)2, py,
CH2Cl2, then MeOH, py; d) FeCl3, 4-Å MS, CH2Cl2; e) BnBr, K2CO3, acetone; f) LiAlH4, THF; g) NaI, TMSCl, MeCN–1,4-dioxane; h) di-
methyl L-glutamate hydrochloride [to give (S)-13] dimethyl D-glutamate hydrochloride [to give (R)-13], K2CO3, MeCN–1,4-dioxane; i) AcOH,
MeOH; j) TFA, aq acetone; k) (COCl)2, DMF, CH2Cl2, then SnCl4, CH2Cl2; l) NaBH4, EtOH; m) TESH, BF3·Et2O, CH2Cl2; n) LiAlH4, THF;
o) H2, 10% Pd/C, HCO2H, MeOH.
Table 1 In Vitro Cytotoxicities of 13a-(S)-Deoxytylophorinine, Its Metabolites, and Its Derivatives Against Cancer Cell Lines
Compound
IC50a (nM)
HepG2
A375
SH-SY5Y
SKOV3
U251
(S)-1
160 ± 30
>5000
510 ± 380
>5000
160 ± 90
>5000
100 ± 30
510 ± 360
150 ± 80
>5000
850 ± 300
>5000
(R)-1
(S)-2
650 ± 450
>5000
1010 ± 280
>5000
1140 ± 840
>5000
1080 ± 670
>5000
(R)-2
(S)-3
2270 ± 1480
2800 ± 670
0.011 ± 0.012
2510 ± 1080
2030 ± 990
>5000
2100 ± 1110
2400 ± 150
0.0054 ± 0.0073
3530 ± 1980
3190 ± 2520
>5000
2270 ± 480
2800 ± 900
0.011 ± 0.021
2510 ± 450
2030 ± 1150
>5000
190 ± 110
140 ± 50
0.00020 ± 0.00015
430 ± 290
360 ± 60
>5000
740 ± 580
1560 ± 1010
0.011 ± 0.0094
1040 ± 900
2680 ± 990
>5000
(R)-3
(S)-4
(R)-4
(S)-5
(R)-5
doxorubicin
paclitaxel
53 ± 45
250 ± 190
480 ± 250
280 ± 120
21 ± 13
>5000
320 ± 170
4.5 ± 3.6
28 ± 33
13 ± 28
a The IC50 values were all measured after 72 h treatment. Each value represents the mean value ± the standard deviation of three independent
experiments performed in triplicate.
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2012, 44, 3757–3764

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PAPER
6-O-Desmethyl-13a-(S)-deoxytylophorinine [(S)-2] and
Most of the synthesized compounds presented marked cy-
totoxic activities in vitro, except for (R)-2 and (R)-5. Inter-
estingly, compounds with an S-configuration at C-13a
showed greater potencies than their enantiomers. There-
fore, the stereochemistry at C-13a plays a fundamental
role in the biological activity. However, the difference in
activity between each pair of enantiomers also depended
on the substituents present on the phenanthrene ring. For
example, (S)-3, the C-7 desmethyl metabolite of (S)-1,
displayed only a slightly more potent cytotoxicity than its
enantiomer (R)-3 against human cancer cell lines other
than SKOV3. However, the C-3 desmethyl metabolite
(S)-4 showed a cytotoxic activity that was five orders of
magnitude greater than that of its enantiomer. Moreover,
(S)-4 showed a much higher cytotoxicity than the parent
compound, or even the positive controls doxorubicin and
paclitaxel. This was probably because the hydroxy group
at C-3 is capable of forming hydrogen bonds with the ac-
tive site of the biological target. The two new C-3-benzyl-
oxy derivatives (S)-5 and (R)-5 had low activities,
presumably due to their lack of planarity and to steric hin-
drance.
7-O-desmethyl-13a-(S)-deoxytylophorinine [(S)-3]; Typical
Procedure
A mixture of Mg powder (114 mg, 1.14 mmol), I2 (560 mg, 1.14
mmol), and anhyd Et2O (20 mL) was refluxed under argon with stir-
ring for 0.5 h in darkness. The mixture was then cooled to r.t. The
clear soln was added to a soln of (+)-13a-(S)-deoxytylophorinine
[(S)-1; 200 mg, 0.55 mmol] in anhyd CH2Cl2 (20 mL), and the mix-
ture was stirred for 0.5 h. The solvent was removed under reduced
pressure, and the residue was heated at 80 °C for 0.5 h in darkness.
AcOH (5 mL), sat. aq Na2S2O3 (20 mL), and 10% MeOH–CH2Cl2
(50 mL) were added and the mixture was stirred for 1 h then filtered.
The filter cake was washed with 10% MeOH–CH2Cl2 (3 × 30 mL).
The organic layer was separated, dried (Na2SO4), filtered, and con-
centrated in vacuo. The residue was purified by flash column chro-
matography [silica gel, CH2Cl2–MeOH (80:1 to 30:1)] to give (S)-2
and (S)-3.
(S)-2
Pale-yellow solid; yield: 58 mg (30%); mp 211.5–212.7 °C (dec.);
[α]D24 +98.0 (c 0.1, CHCl3); >99% ee [flow rate 1.0 mL/min, 18%
i-PrOH–hexane (0.2% Et3N), tR = 16.59 min]; purity: >99%
(HPLC) [flow rate 1.0 mL/min, 32% MeCN–H2O (0.2% Et3N)].
1H NMR (500 MHz, pyridine-d5): δ = 11.59 (s, 1 H), 8.68 (s, 1 H),
8.26 (d, J = 1.1 Hz, 1 H), 8.04 (d, J = 9.0 Hz, 1 H), 7.50 (s, 1 H),
7.43 (dd, J = 9.0, 1.1 Hz, 1 H), 4.85 (d, J = 14.7 Hz, 1 H), 3.96 (s, 3
H), 3.90 (s, 3 H), 3.73 (d, J = 14.7 Hz, 1 H), 3.43–3.34 (m, 2 H),
3.04–2.91 (m, 1 H), 2.47–2.39 (m, 1 H), 2.38–2.30 (m, 1 H), 2.14–
2.06 (m, 1 H), 1.96–1.82 (m, 1 H), 1.80–1.63 (m, 2 H).
1H NMR (600 MHz, DMSO-d6): δ = 9.36 (s, 1 H), 8.03 (s, 1 H),
7.89 (d, J = 9.0 Hz, 1 H), 7.85 (d, J = 1.8 Hz, 1 H), 7.18 (s, 1 H),
7.17 (dd, J = 9.0, 1.8 Hz, 1 H), 4.53 (d, J = 15.0 Hz, 1 H), 3.95 (s, 3
H), 3.94 (s, 3 H), 3.51–3.49 (m, 1 H), 3.33–3.32 (m, 2 H), 2.77–2.72
(m, 1 H), 2.35–2.31 (m, 2 H), 2.14–2.13 (m, 1 H), 1.87–1.81 (m, 2
H), 1.64–1.60 (m, 1 H).
13C NMR (125 MHz, pyridine-d5): δ = 158.7, 150.7, 148.3, 131.5,
127.1, 127.0, 126.6, 126.1, 125.9, 125.4, 116.6, 109.7, 105.1, 104.5,
61.0, 56.2, 55.7 (2 × C), 54.8, 34.5, 32.1, 22.5.
HRMS (ESI): m/z [M + H]+ calcd for C22H24NO3: 350.1751; found:
350.1760.
In conclusion, three optically pure compounds [3-O-des-
methyl-13a-(S)-deoxytylophorinine, 6-O-desmethyl-13a-
(S)-deoxytylophorinine, and 7-O-desmethyl-13a-(S)-de-
oxytylophorinine] and their enantiomers were synthesized
stereospecifically. By comparing the retention times of
the three purified metabolites with those of the synthetic
enantiomers in chiral HPLC, we confirmed that the con-
figuration at C-13a remains unchanged during metabo-
lism. A subsequent biological evaluation demonstrated
that the C-3 desmethyl metabolite of (S)-1 possessed ex-
cellent cytotoxic activities against five human cancer cell
lines. This showed that the presence of a hydrogen-bond
donor at C-3 increased the potency of the lead compound.
This research further underlines the importance of study-
ing the metabolism of natural products for clues to new
drug candidates.
(S)-3
Pale-green solid; yield: 30 mg (16%); mp 172.7–173.9 °C (dec.);
[α]D24 +93.0 (c 0.1, CHCl3); >99% ee [flow rate 1.0 mL/min, 18%
i-PrOH–hexane (0.2% Et3N), tR = 16.61 min]; purity: >99%
(HPLC) [flow rate 1.0 mL/min, 32% MeCN–H2O (0.2% Et3N)].
All materials and reagents were obtained from commercial sources
and used without further purification unless otherwise stated. THF
was distilled from Na and benzophenone, MeCN was distilled from
4 Å MS, and CH2Cl2 was distilled from P2O5. All these distillations
were performed under dry N2, immediately before the solvent was
used. Melting points were determined on an XT5B micromelting
point apparatus (Beijing Keyi Electric Light Instrument Factory,
Beijing) and are uncorrected. NMR spectra were recorded in
CDCl3, DMSO-d6 or pyridine-d5 on a Varian INOVA 500 MHz
spectrometer equipped with a cold probe. Chemical shifts (δ) are re-
ported in ppm. The coupling constants (J) are reported in Hz. High-
resolution mass spectra were recorded with an Agilent Technolo-
gies 6250 Accurate-Mass Q-TOF LC/MS spectrometer. Optical ro-
tations were measured at r.t. with a PerkinElmer 341 MC
polarimeter at the Na D line. Purities and optical purities of the syn-
thesized compounds were assessed by HPLC on Xtimate C18 (4.6 ×
250 mm, 5 μm) or Chiralpak AD-H columns (4.6 × 250 mm, 5 μm),
respectively.
1H NMR (500 MHz, pyridine-d5): δ = 11.62 (br s, 1 H), 8.41 (s, 1
H), 8.39 (s, 1 H), 8.09–8.03 (m, 1 H), 7.81 (s, 1 H), 7.44 (d, J = 8.9
Hz, 1 H), 4.69 (d, J = 14.8 Hz, 1 H), 4.00 (s, 3 H), 3.93 (s, 3 H),
3.66–3.58 (m, 1 H), 3.42–3.30 (m, 2 H), 3.03–2.91 (m, 1 H), 2.42–
2.33 (m, 1 H), 2.32–2.24 (m, 1 H), 2.14–2.03 (m, 1 H), 1.94–1.82
(m, 1 H), 1.78–1.62 (m, 2 H).
13C NMR (125 MHz, pyridine-d5): δ = 158.8, 149.5, 149.2, 132.0,
127.8, 127.6, 126.7, 126.3, 126.2, 124.4, 115.8, 109.1, 105.6, 105.5,
60.9, 56.4, 55.9, 55.7, 54.9, 34.5, 32.1, 22.4.
HRMS (ESI): m/z [M + H]+ calcd for C22H24NO3: 350.1751; found:
350.1746.
6-O-Desmethyl-13a-(R)-deoxytylophorinine [(R)-2] and
7-O-Desmethyl-13a-(R)-deoxytylophorinine [(R)-3]
These compounds were prepared in a similar manner, starting from
(R)-1.
(R)-2
Pale-yellow solid; yield: 52 mg (26%); mp 221.2–222.8 °C (dec.);
[α]D24 –113.0 (c 0.1, CHCl3); >99% ee [flow rate 1.0 mL/min, 18%
i-PrOH–hexane (0.2% Et3N), tR (minor) = 16.58 min, tR (major)
Synthesis 2012, 44, 3757–3764
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PAPER
Monodesmethyl Metabolites of (+)-13a-(S)-Deoxytylophorinine
3761
= 19.41 min]; purity: >99% (HPLC) [flow rate 1.0 mL/min, 32%
MeCN–H2O (0.2% Et3N)].
13C NMR (125 MHz, DMSO-d6): δ = 168.8, 158.1, 148.7, 148.1,
138.5, 132.0 (2 × C), 130.3, 128.9, 126.6, 121.7, 115.1 (2 × C),
113.1, 111.9, 69.1, 55.5, 55.4, 21.7.
HRMS (ESI): m/z [M – H]– calcd for C20H21O5: 341.1394; found:
341.1397.
1H NMR (500 MHz, pyridine-d5): δ = 11.57 (s, 1 H), 8.69 (s, 1 H),
8.26 (d, J = 1.7 Hz, 1 H), 8.05 (d, J = 9.0 Hz, 1 H), 7.51 (s, 1 H),
7.43 (dd, J = 9.0, 1.7 Hz, 1 H), 4.86 (d, J = 14.7 Hz, 1 H), 3.96 (s, 3
H), 3.90 (s, 3 H), 3.74 (d, J = 14.6 Hz, 1 H), 3.43–3.36 (m, 2 H),
3.04–2.94 (m, 1 H), 2.48–2.39 (m, 1 H), 2.38–2.31 (m, 1 H), 2.16–
2.06 (m, 1 H), 1.96–1.84 (m, 1 H), 1.80–1.64 (m, 2 H).
Methyl (2E)-2-(3,4-Dimethoxyphenyl)-3-(4-isopropoxyphe-
nyl)acrylate (9)
Oxalyl chloride (45.90 mL, 0.53 mol) was added in portions to a
soln of acid 8 (120.00 g, 0.35 mol) in CH2Cl2 (500 mL) at r.t. Pyri-
dine (8.70 mL, 0.11 mol) was then added and the mixture was
stirred for 1 h. The solvent was removed under reduced pressure and
the residue was treated with pyridine (43.30 mL, 0.53 mol) and
MeOH (150 mL) at 0 °C, then kept at r.t. for 0.5 h. The mixture was
filtered, washed with MeOH (3 × 30 mL), and concentrated in vac-
uo to give a white solid; yield: 108.93 g (87%); mp 113.9–115.1 °C.
1H NMR (500 MHz, CDCl3): δ = 7.77 (s, 1 H), 7.01 (d, J = 8.8 Hz,
2 H), 6.91 (d, J = 8.2 Hz, 1 H), 6.80 (dd, J = 8.2, 1.8 Hz, 1 H), 6.75
(d, J = 1.7 Hz, 1 H), 6.67 (d, J = 8.8 Hz, 2 H), 4.51 (hept, J = 5.9 Hz,
1 H), 3.94 (s, 3 H), 3.82 (s, 3 H), 3.79 (s, 3 H), 1.30 (d, J = 6.1 Hz,
6 H).
13C NMR (125 MHz, CDCl3): δ = 169.0, 159.0, 149.3, 148.7, 140.5,
132.7 (2 × C), 129.5, 128.9, 127.1, 122.3, 115.5 (2 × C), 113.0,
111.6, 70.0, 56.1, 56.0, 52.5, 22.2 (2 × C).
HRMS (ESI): m/z [M + Na]+ calcd for C21H24NaO5: 379.1516;
found: 379.1528.
13C NMR (125 MHz, pyridine-d5): δ = 158.7, 150.7, 148.3, 131.5,
127.1, 127.0, 126.6, 126.1, 125.9, 125.4, 116.6, 109.7, 105.1, 104.5,
61.0, 56.2, 55.7 (2 × C), 54.8, 34.5, 32.1, 22.5.
HRMS (ESI): m/z [M + H]+ calcd for C22H24NO3: 350.1751; found:
350.1757.
(R)-3
Pale-green solid; yield: 26 mg (14%); mp 177.6–178.9 °C (dec.);
[α]D24 –91.0 (c 0.1, CHCl3); >99% ee [flow rate 1.0 mL/min, 18%
i-PrOH–hexane (0.2% Et3N), tR (minor) = 16.22 min, tR (major) =
32.98 min]; purity: >99% (HPLC) [flow rate 1.0 mL/min, 32%
MeCN–H2O (0.2% Et3N)].
1H NMR (500 MHz, pyridine-d5): δ = 11.63 (br s, 1 H), 8.42 (s, 1
H), 8.40 (d, J = 2.4 Hz, 1 H), 8.06 (d, J = 9.0 Hz, 1 H), 7.80 (s, 1 H),
7.44 (dd, J = 9.0, 2.4 Hz, 1 H), 4.71 (d, J = 14.8 Hz, 1 H), 4.00 (s, 3
H), 3.93 (s, 3 H), 3.76–3.71 (m, 1 H), 3.43–3.32 (m, 2 H), 3.03–2.92
(m, 1 H), 2.45–2.35 (m, 1 H), 2.34–2.26 (m, 1 H), 2.15–2.04 (m, 1
H), 1.95–1.82 (m, 1 H), 1.79–1.63 (m, 2 H).
13C NMR (125 MHz, pyridine-d5): δ = 158.8, 149.5, 149.2, 132.0,
127.7, 127.6, 126.3, 126.2 (2 × C), 124.4, 115.8, 109.1, 105.6,
105.5, 61.0, 56.4, 55.9, 55.7, 54.8, 32.1, 30.5, 22.4.
HRMS (ESI): m/z [M + H]+ calcd for C22H24NO3: 350.1751; found:
350.1757.
Methyl 3-(Benzyloxy)-6,7-dimethoxyphenanthrene-9-carboxyl-
ate (11)
Ester 9 (10.00 g, 0.03 mol) was added to a mixture of anhyd FeCl3
(18.58 g, 0.11 mol), 4 Å MS (30 g), and CH2Cl2 (300 mL) at 0 °C,
and the mixture was stirred for 8 h. The reaction was then quenched
with sat. aq NaHCO3 (150 mL) and filtered. The organic layer was
collected, dried (Na2SO4), filtered, and concentrated. The crude hy-
droxy ester 10 was used in the next step without further purification.
4-Isopropoxybenzaldehyde (7)
A mixture of 4-hydroxybenzaldehyde (6; 100.00 g, 0.80 mol),
i-PrBr (115.34 mL, 1.20 mol), anhyd K2CO3 (169.80 g, 1.20 mol),
anhyd KI (13.52 g, 0.08 mol), and DMF (500 mL) was stirred at
55 °C for 6 h. A second portion of i-PrBr (38.45 mL, 0.40 mol) was
then added and the mixture was stirred for a further 12 h at 55 °C,
cooled to r.t., diluted with H2O (200 mL), and filtered. The aqueous
phase was extracted with EtOAc (3 × 150 mL). The organic phases
were combined and washed sequentially with 10% aq NaOH, H2O,
and brine then dried (Na2SO4), filtered, and concentrated under re-
duced pressure to give a pale-yellow oil; yield: 128.30 g (97%).
A mixture of crude hydroxy ester 10, BnBr (5.20 mL, 0.04 mol),
K2CO3 (7.91 g, 0.06 mol), and acetone (100 mL) was refluxed for 3
h, cooled, diluted with H2O (100 mL), and extracted with CH2Cl2 (3
× 100 mL). The combined organic layers were dried (Na2SO4), fil-
tered, and concentrated. The residue was purified by flash column
chromatography [silica gel, PE–EtOAc–CH2Cl2 (20:4:1)] to give 11
as a pale-yellow solid; yield: 5.91 g (52%); mp 133.8–134.9 °C.
1H NMR (500 MHz, CDCl3): δ = 8.66 (s, 1 H), 8.46 (s, 1 H), 7.92
(d, J = 2.0 Hz, 1 H), 7.88 (d, J = 8.8 Hz, 1 H), 7.82 (s, 1 H), 7.57–
7.54 (m, 2 H), 7.47–7.42 (m, 2 H), 7.40–7.36 (m, 1 H), 7.31 (dd,
J = 8.8, 2.3 Hz, 1 H), 5.32 (s, 2 H), 4.11 (s, 3 H), 4.09 (s, 3 H), 4.03
(s, 3 H).
1H NMR (500 MHz, CDCl3): δ = 9.85 (s, 1 H), 7.80 (d, J = 8.7 Hz,
2 H), 6.96 (d, J = 8.7 Hz, 2 H), 4.66 (hept, J = 6.0 Hz, 1 H), 1.36 (d,
J = 6.1 Hz, 6 H).
13C NMR (125 MHz, CDCl3): δ = 190.9, 163.3, 132.2 (2 × C),
129.7, 115.7, 70.4, 22.0.
HRMS (ESI): m/z [M + Na]+ calcd for C10H12NaO2: 187.0730;
found: 187.0729.
13C NMR (125 MHz, CDCl3): δ = 168.4, 159.6, 150.1, 149.1, 136.9,
133.6, 132.1, 131.4, 129.0 (2 × C), 128.5, 127.8 (2 × C), 125.3,
125.2, 124.6, 122.0, 116.6, 107.1, 105.6, 103.4, 70.7, 56.1 (2 × C),
52.3.
HRMS (ESI): m/z [M + Na]+ calcd for C25H22NaO5: 425.1359;
found: 425.1362.
(2E)-2-(3,4-Dimethoxyphenyl)-3-(4-isopropoxyphenyl)acrylic
Acid (8)
A mixture of aldehyde 7 (50.00 g, 0.80 mol), (3,4-dimethoxyphe-
nyl)acetic acid (57.47 g, 0.29 mol), Et3N (49.00 mL, 0.35 mol), and
Ac2O (70.00 mL, 0.73 mol) was refluxed with stirring under argon
for 8 h, then cooled to r.t. The mixture was diluted with EtOAc (500
mL) and washed with H2O (3 × 300 mL). The organic phase was
dried, filtered, and concentrated under reduced pressure to give a
pale-yellow solid; yield: 70.35 g (69%); mp 164.0–165.2 °C.
1H NMR (500 MHz, DMSO-d6): δ = 12.40 (br s, 1 H), 7.65 (s, 1 H),
7.03 (d, J = 8.6 Hz, 2 H), 6.96 (d, J = 8.2 Hz, 1 H), 6.79–6.71 (m, 3
H), 6.67 (d, J = 8.2 Hz, 1 H), 4.63–4.54 (m, 1 H), 3.78 (s, 3 H), 3.66
(s, 3 H), 1.21 (d, J = 6.0 Hz, 6 H).
[3-(Benzyloxy)-6,7-dimethoxy-9-phenanthryl]methanol (12)
A soln of ester 11 (19.76 g, 49.10 mmol) in THF (300 mL) at 0 °C
was added dropwise to a suspension of LiAlH4 (3.80 g, 98.20 mmol)
in THF (20 mL), and the mixture was stirred for 0.5 h at 0 °C. The
reaction was then quenched with H2O (20 mL) and 10% aq HCl
(200 mL). The mixture was filtered and the filtrate was extracted
with CH2Cl2 (3 × 200 mL). The combined organic phase was dried,
filtered, and concentrated to give a white powder; yield: 17.85 g
(97%); mp 190.7–191.5 °C.
1H NMR (500 MHz, CDCl3): δ = 7.95 (d, J = 1.9 Hz, 1 H), 7.87 (s,
1 H), 7.80 (d, J = 8.7 Hz, 1 H), 7.63 (s, 1 H), 7.58–7.53 (m, 3 H),
7.46–7.41 (m, 2 H), 7.39–7.35 (m, 1 H), 7.30–7.28 (m, 1 H), 5.30
(s, 2 H), 5.13 (s, 2 H), 4.11 (s, 3 H), 4.08 (s, 3 H).
© Georg Thieme Verlag Stuttgart · New York
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P. Yu et al.
PAPER
13C NMR (125 MHz, CDCl3): δ = 157.8, 149.7, 149.1, 137.2, 131.7,
131.5, 130.4, 128.9 (2 × C), 128.4, 127.9 (2 × C), 126.0, 125.9,
125.1, 124.8, 116.1, 105.9, 105.1, 104.0, 70.7, 65.0, 56.2 (2 × C).
HRMS (ESI): m/z [M + Na]+ calcd for C24H22NaO4: 397.1410;
found: 397.1413.
was then maintained at reflux for an additional 2 h. The mixture was
then cooled to r.t. and 1 M HCl (150 mL) was added. The organic
layer was separated, washed with H2O (3 × 50 mL), dried (Na2SO4),
filtered, and concentrated in vacuo. The residue was purified by
flash column chromatography [silica gel, CH2Cl2–MeOH (100:1)]
to give a pale-yellow solid; yield: 3.10 g (84%); mp 209.7–
210.9 °C; [α]D24 +117.3 (c 0.3, CHCl3).
1H NMR (500 MHz, CDCl3): δ = 9.36 (d, J = 9.4 Hz, 1 H), 7.94 (d,
J = 2.5 Hz, 1 H), 7.81 (s, 1 H), 7.57–7.54 (m, 2 H), 7.46–7.41 (m, 2
H), 7.40–7.35 (m, 2 H), 7.31 (s, 1 H), 5.73 (d, J = 17.9 Hz, 1 H),
5.30 (s, 2 H), 4.68 (d, J = 17.6 Hz, 1 H), 4.48–4.40 (m, 1 H), 4.13
(s, 3 H), 4.09 (s, 3 H), 2.68–2.48 (m, 4 H).
13C NMR (125 MHz, CDCl3): δ = 195.5, 174.3, 157.7, 152.0, 150.3,
137.4, 137.1, 131.4, 129.4, 129.0 (2 × C), 128.5, 128.1, 127.8 (2 ×
C), 123.5, 123.0, 122.7, 116.8, 106.4, 104.6, 104.0, 70.7, 61.5, 56.5,
56.3, 41.0, 30.3, 21.0.
HRMS (ESI): m/z [M + Na]+ calcd for C29H25NNaO5: 490.1625;
found: 490.1617.
Methyl (2S)-1-{[3-(Benzyloxy)-6,7-dimethoxy-9-phenan-
thryl]methyl}-5-oxopyrrolidine-2-carboxylate [(S)-13]
A mixture of alcohol 12 (9.97 g, 26.63 mmol), NaI (8.14 g, 53.25
mmol), 1,4-dioxane (500 mL), and MeCN (250 mL) was stirred at
60 °C for 0.5 h. The mixture was then cooled to r.t., treated with
TMSCl (5.17 mL, 39.94 mmol), and stirred at r.t. for 1 h. Dimethyl
L-glutamate hydrochloride (8.45 g, 39.94 mmol) and anhyd K2CO3
(11.26 g, 79.88 mmol) were added and the mixture was stirred for
another 8 h. The mixture was then partitioned between H2O (200
mL) and CH2Cl2 (200 mL). The organic layer was separated, dried
(Na2SO4), filtered, and concentrated in vacuo. The residue was di-
rectly purified by flash column chromatography [silica gel, PE–
EtOAc (3:1)] to give a pale-yellow oil to which AcOH (50 mL) and
MeOH (100 mL) were quickly added. The resulting mixture was
stirred at 60 °C for 4 h then concentrated in vacuo. The residue was
washed with Et2O (3 × 5 mL), filtered, and dried to give a white
powder; yield: 10.84 g (82%); mp 172.4–173.7 °C; [α]D24 +54.8 (c
1.0, CHCl3).
(13aS)-3-(Benzyloxy)-6,7-dimethoxy-12,13,13a,14-tetrahydro-
dibenzo[f,h]pyrrolo[1,2-b]isoquinolin-11(9H)-one [(S)-16]
NaBH4 (1.38 g, 35.77 mmol) was added to a stirred soln of (S)-15
(8.36 g, 17.88 mmol) in a mixture of CH2Cl2 (150 mL) and MeOH
(150 mL), and the resulting mixture was stirred for 15 min. The re-
action was then quenched with sat. aq NH4Cl (150 mL). The organic
layer was separated, dried (Na2SO4), filtered, and concentrated in
vacuo. The residue was dissolved in CH2Cl2 (100 mL), and TESH
(5.80 mL, 35.77 mmol) and BF3·Et2O (6.83 mL, 53.68 mmol) were
added. The mixture was stirred for another 4 h and then sat. aq
NaHCO3 (60 mL) was added. The organic layer was separated,
dried (Na2SO4), filtered, and concentrated in vacuo. The residue
was purified by flash column chromatography [silica gel, CH2Cl2–
MeOH (100:1)] to give a pale-yellow solid; yield: 7.54 g (93%); mp
237.8–238.7 °C; [α]D24 +187.0 (c 1.0, CHCl3).
1H NMR (500 MHz, CDCl3): δ = 7.99 (d, J = 2.3 Hz, 1 H), 7.94 (d,
J = 9.1 Hz, 1 H), 7.85 (s, 1 H), 7.57–7.53 (m, 2 H), 7.46–7.41 (m, 2
H), 7.39–7.35 (m, 1 H), 7.32 (dd, J = 9.0, 2.4 Hz, 1 H), 7.19 (s, 1 H),
5.33 (d, J = 17.0 Hz, 1 H), 5.31 (s, 2 H), 4.57 (d, J = 17.0 Hz, 1 H),
4.10 (s, 3 H), 4.07 (s, 3 H), 4.00–3.91 (m, 1 H), 3.59–3.53 (m, 1 H),
2.92–2.84 (m, 1 H), 2.66–2.60 (m, 2 H), 2.59–2.51 (m, 1 H), 2.08–
1.97 (m, 1 H).
1H NMR (500 MHz, CDCl3): δ = 7.93 (d, J = 2.1 Hz, 1 H), 7.84 (s,
1 H), 7.76 (d, J = 8.7 Hz, 1 H), 7.63 (s, 1 H), 7.57–7.53 (m, 2 H),
7.47–7.41 (m, 3 H), 7.39–7.35 (m, 1 H), 7.30–7.27 (m, 1 H), 5.51
(d, J = 14.6 Hz, 1 H), 5.30 (s, 2 H), 4.41 (d, J = 14.5 Hz, 1 H), 4.10
(s, 3 H), 4.05 (s, 3 H), 3.84 (dd, J = 9.3, 3.3 Hz, 1 H), 3.59 (s, 3 H),
2.66–2.56 (m, 1 H), 2.44–2.36 (m, 1 H), 2.20–2.07 (m, 1 H), 2.03–
1.95 (m, 1 H).
13C NMR (125 MHz, CDCl3): δ = 174.8, 172.4, 158.0, 150.0, 149.2,
137.2, 131.7, 130.3, 129.0 (2 × C), 128.4, 127.8 (2 × C), 127.7,
126.8, 126.1, 125.6, 124.9, 116.2, 105.9, 105.5, 103.8, 70.8, 58.7,
56.6, 56.2, 52.5, 45.0, 30.1, 23.0.
HRMS (ESI): m/z [M + Na]+ calcd for C30H29NNaO6: 522.1887;
found: 522.1898.
(2S)-1-{[3-(Benzyloxy)-6,7-dimethoxy-9-phenanthryl]methyl}-
5-oxopyrrolidine-2-carboxylic Acid [(S)-14]
A mixture of ester (S)-13 (1.18 g, 2.36 mol), TFA (5.40 mL, 120.10
mmol), H2O (5 mL), and acetone (13 mL) was stirred at 60 °C for
12 h. The solvent was removed under reduced pressure and the res-
idue was purified by flash column chromatography [silica gel,
CH2Cl2–MeOH (100:1)] to give a white solid; yield: 1.15 g (90%);
mp 253.4–254.3 °C; [α]D24 +46.3 (c 1.0, DMF).
13C NMR (125 MHz, CDCl3): δ = 174.5, 157.3, 150.0, 149.0, 137.2,
130.9, 129.0 (2 × C), 128.4, 127.8 (2 × C), 125.4, 125.2, 125.1,
125.0, 123.7, 122.5, 115.8, 106.6, 104.1, 103.1, 70.8, 56.4, 56.2,
53.6, 41.4, 33.5, 30.4, 25.5.
HRMS (ESI): m/z [M + Na]+ calcd for C29H27NNaO4: 476.1832;
found: 476.1836.
1H NMR (500 MHz, DMSO-d6): δ = 13.01 (s, 1 H), 8.17 (s, 1 H),
8.07 (s, 1 H), 7.84 (d, J = 8.7 Hz, 1 H), 7.60–7.56 (m, 2 H), 7.52 (s,
1 H), 7.49 (s, 1 H), 7.45–7.40 (m, 2 H), 7.35 (t, J = 7.2 Hz, 1 H),
7.29 (dd, J = 8.7, 1.6 Hz, 1 H), 5.39 (d, J = 14.6 Hz, 1 H), 5.37 (s, 2
H), 4.23 (d, J = 14.6 Hz, 1 H), 4.03 (s, 3 H), 3.88 (s, 3 H), 3.67 (dd,
J = 9.2, 3.2 Hz, 1 H), 2.46–2.37 (m, 1 H), 2.37–2.28 (m, 1 H), 2.18–
2.07 (m, 1 H), 1.93–1.83 (m, 1 H).
13C NMR (125 MHz, DMSO-d6): δ = 174.2, 173.2, 157.3, 149.4,
148.9, 137.2, 130.9, 130.0, 128.5 (2 × C), 128.0 (2 × C), 127.9,
126.6, 126.3, 125.4, 124.9, 124.4, 116.3, 105.5, 105.0, 104.6, 69.7,
57.9, 55.9, 55.5, 43.6, 29.3, 22.3.
(13aS)-3-(Benzyloxy)-6,7-dimethoxy-9,11,12,13,13a,14-hexahy-
drodibenzo[f,h]pyrrolo[1,2-b]isoquinoline [(S)-5]
LiAlH4 (88 mg, 2.28 mmol) was added to a suspension of ketone
(S)-16 (520 mg, 1.14 mmol) in THF (50 mL), and the mixture was
refluxed with stirring in darkness for 2 h. The mixture was then al-
lowed to cool to r.t. and the reaction was quenched with acetone (1
mL) and H2O (20 mL). The mixture was filtered, dried (Na2SO4),
and concentrated in vacuo. The residue was purified by flash col-
umn chromatography [silica gel, CH2Cl2–MeOH (60:1)] to give a
24
white solid; yield: 421 mg (84%); mp 205.5–206.6 °C (dec.); [α]D
HRMS (ESI): m/z [M – H]– calcd for C29H26NO6: 484.1766; found:
484.1770.
+101.0 (c 1.0, CHCl3); 99% ee [flow rate 1.0 mL/min, 22%
i-PrOH–hexane (0.2% Et3N), tR = 30.25 min]; purity: >99%
(HPLC) [flow rate 1.0 mL/min, 40% MeCN–H2O (0.2% Et3N)].
1H NMR (500 MHz, pyridine-d5): δ = 8.53 (d, J = 1.6 Hz, 1 H), 8.36
(s, 1 H), 8.09 (d, J = 9.0 Hz, 1 H), 7.65 (d, J = 7.6 Hz, 2 H), 7.57–
7.54 (m, 1 H), 7.50–7.42 (m, 3 H), 7.37 (t, J = 7.3 Hz, 1 H), 5.40 (s,
2 H), 4.83 (d, J = 14.7 Hz, 1 H), 4.02 (s, 3 H), 3.97 (s, 3 H), 3.72 (d,
J = 14.8 Hz, 1 H), 3.45–3.36 (m, 2 H), 3.05–2.92 (m, 1 H), 2.47–
(13aS)-3-(Benzyloxy)-6,7-dimethoxy-13,13a-dihydrodiben-
zo[f,h]pyrrolo[1,2-b]isoquinoline-11,14(9H,12H)-dione [(S)-15]
Oxalyl chloride (0.86 mL, 9.94 mmol) and DMF (0.30 mL) were
added to a soln of acid (S)-14 (3.84 g, 7.91 mmol) in anhyd CH2Cl2
(200 mL). The mixture was stirred at r.t. for 1 h then heated to re-
flux. A soln of SnCl4 (2.35 mL, 19.80 mmol)) in anhyd CH2Cl2 (50
mL) was added in five portions, one every hour, and the mixture
Synthesis 2012, 44, 3757–3764
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PAPER
Monodesmethyl Metabolites of (+)-13a-(S)-Deoxytylophorinine
3763
2.38 (m, 1 H), 2.38–2.31 (m, 1 H), 2.18–2.06 (m, 1 H), 1.96–1.84
(m, 1 H), 1.81–1.65 (m, 2 H).
13C NMR (125 MHz, pyridine-d5): δ = 158.0, 151.0, 149.9, 138.6,
131.6, 129.4 (2 × C), 128.8, 128.7 (2 × C), 128.1, 127.0, 126.8,
126.7, 126.3, 124.6, 116.7, 107.1, 105.8, 104.9, 71.0, 60.9, 56.5,
56.3, 55.7, 54.8, 34.6, 32.1, 22.5.
1H NMR (500 MHz, DMSO-d6): δ = 12.99 (br s, 1 H), 8.17 (s, 1 H),
8.07 (s, 1 H), 7.84 (d, J = 8.7 Hz, 1 H), 7.60–7.56 (m, 2 H), 7.52 (s,
1 H), 7.49 (s, 1 H), 7.46–7.40 (t, J = 7.5 Hz, 2 H), 7.35 (t, J = 7.3
Hz, 1 H), 7.29 (dd, J = 8.7, 1.7 Hz, 1 H), 5.30 (d, J = 14.7 Hz, 1 H),
5.37 (s, 2 H), 4.23 (d, J = 14.7 Hz, 1 H), 4.03 (s, 3 H), 3.88 (s, 3 H),
3.67 (dd, J = 9.3, 3.2 Hz, 1 H), 2.46–2.37 (m, 1 H), 2.37–2.28 (m, 1
H), 2.18–2.10 (m, 1 H), 1.92–1.84 (m, 1 H).
13C NMR (125 MHz, DMSO-d6): δ = 174.2, 173.2, 157.3, 149.4,
148.9, 137.2, 130.9, 130.0, 128.5 (2 × C), 128.0 (2 × C), 127.9,
126.6, 126.4, 125.4, 124.9, 124.4, 116.3, 105.5, 105.0, 104.6, 69.7,
57.9, 55.9, 55.5, 43.7, 29.3, 22.3.
HRMS (ESI): m/z [M + H]+ calcd for C29H30NO3: 440.2220; found:
440.2244.
3-O-Desmethyl-13a-(S)-deoxytylophorinine [(S)-4]
A mixture of benzyloxy derivative (S)-5 (300 mg, 0.68 mmol), 10%
Pd/C (50 mg), HCO2H (40 mL), and MeOH (20 mL) was stirred at
70 °C in darkness under H2 (1.0 atm) for 12 h. The mixture was then
filtered and concentrated in vacuo. The residue was purified by
flash column chromatography [silica gel, CH2Cl2–MeOH (60:1
then 40:1)] to give a white solid; yield: 188 mg (76%); mp 205.5–
206.9 °C (dec.); [α]D24 +126.6 (c 1.0, DMF); >99% ee [flow rate 1.0
mL/min, 18% i-PrOH–hexane (0.2% Et3N), tR (major) = 14.55 min,
tR (minor) = 31.77 min]; purity: >99% (HPLC) [flow rate 1.0
mL/min, 25% MeCN–H2O (0.2% Et3N)].
HRMS (ESI): m/z [M – H]– calcd for C29H26NO6: 484.1766; found:
484.1770.
(13aR)-3-(Benzyloxy)-6,7-dimethoxy-13,13a-dihydrodiben-
zo[f,h]pyrrolo[1,2-b]isoquinoline-11,14(9H,12H)-dione [(R)-15]
24
Pale-yellow solid; yield: 2.95 g (82%); mp 214.0–215.2 °C; [α]D
–126.3 (c 1.0, CHCl3).
1H NMR (500 MHz, CDCl3): δ = 9.35 (d, J = 9.4 Hz, 1 H), 7.93 (d,
J = 2.4 Hz, 1 H), 7.80 (s, 1 H), 7.56–7.51 (m, 2 H), 7.45–7.40 (m, 2
H), 7.39–7.34 (m, 2 H), 7.30 (s, 1 H), 5.71 (d, J = 17.9 Hz, 1 H),
5.29 (s, 2 H), 4.67 (d, J = 17.9 Hz, 1 H), 4.47–4.39 (m, 1 H), 4.12
(s, 3 H), 4.08 (s, 3 H), 2.66–2.49 (m, 4 H).
13C NMR (125 MHz, CDCl3): δ = 195.5, 174.3, 157.7, 152.0, 150.2,
137.4, 137.1, 131.4, 129.4, 129.0 (2 × C), 128.5, 128.1, 127.8
(2 × C), 123.5, 123.0 (2 × C), 122.7, 116.8, 106.4, 104.6, 104.0,
70.7, 61.5, 56.5, 56.3, 41.0, 30.3, 21.0.
1H NMR (500 MHz, pyridine-d5): δ = 11.84 (s, 1 H), 8.52 (s, 1 H),
8.17 (s, 1 H), 8.09 (d, J = 8.8 Hz, 1 H), 7.63 (d, J = 8.8 Hz, 1 H),
7.45 (s, 1 H), 4.82 (d, J = 14.7 Hz, 1 H), 3.95 (s, 3 H), 3.93 (s, 3 H),
3.72 (d, J = 14.5 Hz, 1 H), 3.45 (d, J = 14.1 Hz, 1 H), 3.39 (t, J = 8.3
Hz, 1 H), 3.07–2.97 (m, 1 H), 2.48–2.39 (m, 1 H), 2.35 (q, J = 8.5
Hz, 1 H), 2.16–2.06 (m, 1 H), 1.96–1.83 (m, 1 H), 1.80–1.63 (m, 2
H).
1H NMR (400 MHz, DMSO-d6): δ = 8.17 (s, 1 H), 7.95 (d, J = 2.0
Hz, 1 H), 7.93 (s, 1 H), 7.84 (d, J = 8.8 Hz, 1 H), 7.18 (s, 1 H), 7.10
(dd, J = 8.8, 2.0 Hz, 1 H), 4.57 (d, J = 15.2 Hz, 1 H), 3.99 (s, 3 H),
3.93 (s, 3 H), 3.56 (d, J = 15.2 Hz, 1 H), 3.38–3.34 (m, 2 H), 2.81–
2.75 (m, 1 H), 2.50–2.39 (m, 2 H), 2.18–2.13 (m, 1 H), 1.91–1.85
(m, 2 H), 1.69–1.62 (m, 1 H).
13C NMR (125 MHz, pyridine-d5): δ = 157.7, 150.8, 149.6, 132.0,
128.2, 126.7, 126.3, 126.0, 125.7, 124.4, 117.7, 108.0, 105.4, 104.9,
61.0, 56.3, 56.1, 55.8, 54.8, 34.6, 32.1, 22.5.
HRMS (ESI): m/z [M + Na]+ calcd for C29H25NNaO5: 490.1625;
found: 490.1628.
(13aR)-3-(Benzyloxy)-6,7-dimethoxy-12,13,13a,14-tetrahy-
drodibenzo[f,h]pyrrolo[1,2-b]isoquinolin-11(9H)-one [(R)-16]
Pale-yellow solid; yield: 4.59 g (91%); mp 236.9–238.0 °C; [α]D
24
–195.0 (c 1.0, CHCl3).
1H NMR (500 MHz, CDCl3): δ = 8.00 (s, 1 H), 7.94 (d, J = 9.0 Hz,
1 H), 7.85 (s, 1 H), 7.55 (d, J = 7.4 Hz, 2 H), 7.46–7.42 (m, 2 H),
7.39–7.35 (m, 1 H), 7.32 (dd, J = 9.1, 2.1 Hz, 1 H), 7.19 (s, 1 H),
5.34 (d, J = 17.0 Hz, 1 H), 5.31 (s, 2 H), 4.57 (d, J = 16.7 Hz, 1 H),
4.10 (s, 3 H), 4.07 (s, 3 H), 4.00–3.92 (m, 1 H), 3.59–3.53 (m, 1 H),
2.94–2.83 (m, 1 H), 2.68–2.50 (m, 3 H), 2.07–1.99 (m, 1 H).
13C NMR (125 MHz, CDCl3): δ = 174.5, 157.3, 150.0, 149.0, 137.2,
130.9, 129.0 (2 × C), 128.4, 127.8 (2 × C), 125.4, 125.2, 125.1,
125.0, 123.7, 122.5, 115.8, 106.6, 104.1, 103.1, 70.7, 56.4, 56.2,
53.6, 41.4, 33.5, 31.2, 25.5.
HRMS (ESI): m/z [M + H]+ calcd for C22H24NO3: 350.1751; found:
350.1758.
Methyl (2R)-1-{[3-(Benzyloxy)-6,7-dimethoxy-9-phenan-
thryl]methyl}-5-oxopyrrolidine-2-carboxylate [(R)-13]
This was prepared in a similar manner to (S)-13, but starting from
dimethyl D-glutamate hydrochloride. Subsequent steps leading to
the synthesis of (R)-4 were identical to those applied to the corre-
sponding enantiomeric compounds.
HRMS (ESI): m/z [M + Na]+ calcd for C29H27NNaO4: 476.1832;
found: 476.1834.
24
White solid; yield: 9.68 g (80%); mp 172.0–174.5 °C (dec.); [α]D
–54.1 (c 1.0, CHCl3).
1H NMR (500 MHz, CDCl3): δ = 7.93 (d, J = 1.8 Hz, 1 H), 7.84 (s,
1 H), 7.76 (d, J = 8.7 Hz, 1 H), 7.63 (s, 1 H), 7.57–7.53 (m, 2 H),
7.47–7.41 (m, 3 H), 7.39–7.35 (m, 1 H), 7.30–7.27 (m, 1 H), 5.51
(d, J = 14.6 Hz, 1 H), 5.30 (s, 2 H), 4.41 (d, J = 14.5 Hz, 1 H), 4.10
(s, 3 H), 4.05 (s, 3 H), 3.84 (dd, J = 9.1, 3.1 Hz, 1 H), 3.59 (s, 3 H),
2.66–2.56 (m, 1 H), 2.47–2.34 (m, 1 H), 2.17–2.06 (m, 1 H), 2.05–
1.93 (m, 1 H).
13C NMR (125 MHz, CDCl3): δ = 174.9, 172.5, 158.0, 150.0, 149.2,
137.2, 131.7, 130.3, 129.0 (2 × C), 128.4, 127.8 (2 × C), 127.7,
126.8, 126.1, 125.6, 124.9, 116.2, 105.9, 105.5, 103.8, 70.8, 58.7,
56.6, 56.2, 52.5, 45.0, 30.1, 23.0.
(13aR)-3-(Benzyloxy)-6,7-dimethoxy-9,11,12,13,13a,14-hexahy-
drodibenzo[f,h]pyrrolo[1,2-b]isoquinoline [(R)-5]
Pale-yellow solid; yield: 215 mg (81%); mp 204.3–205.0 °C (dec.);
[α]D24 –109.9 (c 1.0, CHCl3); >99% ee [flow rate 1.0 mL/min, 22%
i-PrOH–hexane (0.2% Et3N), tR (minor) = 30.26 min, tR (major) =
48.30 min]; purity: >99% (HPLC) [flow rate 1.0 mL/min, 40%
MeCN–H2O (0.2% Et3N)].
1H NMR (500 MHz, pyridine-d5): δ = 8.54 (d, J = 2.4 Hz, 1 H), 8.37
(s, 1 H), 8.09 (d, J = 9.0 Hz, 1 H), 7.65 (d, J = 7.4 Hz, 2 H), 7.56 (d,
J = 2.4 Hz, 1 H), 7.49–7.43 (m, 3 H), 7.40–7.35 (m, 1 H), 5.40 (s, 2
H), 4.84 (d, J = 14.7 Hz, 1 H), 4.02 (s, 3 H), 3.97 (s, 3 H), 3.73 (d,
J = 14.7 Hz, 1 H), 3.45–3.36 (m, 2 H), 3.04–2.95 (m, 1 H), 2.48–
2.40 (m, 1 H), 2.39–2.31 (m, 1 H), 2.17–2.08 (m, 1 H), 1.97–1.85
(m, 1 H), 1.81–1.65 (m, 2 H).
HRMS (ESI): m/z [M + Na]+ calcd for C30H29NNaO6: 522.1887;
found: 522.1899.
13C NMR (125 MHz, pyridine-d5): δ = 158.0, 151.0, 149.9, 138.6,
131.6, 129.4 (2 × C), 128.8, 128.7 (2 × C), 128.1, 127.0, 126.8,
126.7, 126.3, 124.6, 116.7, 107.1, 105.8, 104.9, 71.0, 61.0, 56.5,
56.3, 55.7, 54.8, 34.6, 32.1, 22.5.
(2R)-1-{[3-(Benzyloxy)-6,7-dimethoxy-9-phenanthryl]methyl}-
5-oxopyrrolidine-2-carboxylic Acid [(R)-14]
White solid; yield: 2.15 g (88%); mp 257.7–258.9 °C; [[α]D24 –43.1
(c 1.0, DMF).
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2012, 44, 3757–3764

3764
P. Yu et al.
PAPER
HRMS (ESI): m/z [M + H]+ calcd for C29H30NO3: 440.2220; found:
440.2237.
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White solid; yield: 152 mg (75%); mp 205.0–206.2 °C (dec.); [α]D
–130.4 (c 1.0, DMF); >99% ee [flow rate 1.0 mL/min, 18%
i-PrOH–hexane (0.2% Et3N), tR (minor) = 14.59 min, tR (major)
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Cytotoxicity Assays
A375, SH-SY5Y, HepG2, and SKOV3 cancer cell lines were ob-
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Cell Culture Center, Institute of Basic Medical Sciences, Chinese
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Acknowledgment
This work was performed with financial support from the National
Natural Science Foundation of China (No. 21132009) and from the
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Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synthesis.SnuIomprifgtooatrinSugpIoi
n
nfomartit
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Synthesis 2012, 44, 3757–3764
© Georg Thieme Verlag Stuttgart · New York