Total synthesis of phenanthroindolizidine alkaloids (±)-antofine, (±)-deoxypergularinine, and their dehydro congeners and evaluation of their cytotoxic activity

Article abstract of DOI:10.1016/j.bmc.2008.04.032

Due to their limited natural abundance and significant biochemical effects, we synthesized the alkaloids (±)-antofine (1a), (±)-deoxypergularinine (1b), and their dehydro congeners (2 and 3) starting from the corresponding phenanthrene-9-carboxaldehydes.

Full text of DOI:10.1016/j.bmc.2008.04.032

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 6233–6241
Total synthesis of phenanthroindolizidine alkaloids
( )-antofine, ( )-deoxypergularinine, and their dehydro
congeners and evaluation of their cytotoxic activity
Chung-Ren Su,a Amooru G. Damu,a Po-Cheng Chiang,b Kenneth F. Bastow,b
Susan L. Morris-Natschke,b Kuo-Hsiung Leeb and Tian-Shung Wua,*
aDepartment of Chemistry, National Cheng Kung University, Tainan 701, Taiwan, ROC
bNatural Products Research Laboratories, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, United States
Received 21 September 2007; revised 11 April 2008; accepted 16 April 2008
Available online 18 April 2008
Abstract—Due to their limited natural abundance and significant biochemical effects, we synthesized the alkaloids ( )-antofine (1a),
( )-deoxypergularinine (1b), and their dehydro congeners (2 and 3) starting from the corresponding phenanthrene-9-carboxaldehy-
des. We also evaluated their in vitro cytotoxic activity. Compounds 1a and 1b showed significant potency against various human
tumor cell lines, including a drug-resistant variant, with EC50 values ranging from 0.16 to 16 ng/mL. Structure–activity correlations
of these alkaloids and some of their synthetic intermediates were also ascertained. The non-planar structure between the two major
moieties, phenanthrene and indolizidine, plays a crucial role in the cytotoxic activity of phenanthroindolizidines. Increasing the pla-
narity and rigidity of the indolizidine moiety significantly reduced potency. A methoxy group at the 2-position (1a) was more favor-
able for cytotoxic activity than a hydrogen atom (1b).
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
Due to low natural abundance of this compound type,
various syntheses of 1a and other phenanthroindolizi-
dine alkaloids, in both racemic and optically active
forms, have been reported in an effort to further
investigate the biochemical and pharmaceutical ef-
fects.8–14 The most commonly reported synthetic
methodology toward the highly condensed natural
alkaloid ( )-1a and its geometric isomer ( )-deoxyper-
gularinine (1b) involves formation of the biaryl bond
of the phenanthrene ring system in the final step, after
the coupling of the aromatic residues with the appro-
priate heterocycle. The other common sequence to
these alkaloids uses differentially substituted phenan-
threnes as starting materials, which are coupled with
the appropriate heterocycle through a halomethyl moi-
ety. Diverse strategies have been employed for attach-
ing the pyrrolidine ring system. These methods include
strategies based on the alkylation of pyrrole, alkyl-
ation of proline, use of a pyrrole-substituted Wittig-
like reagent, and ortho metalation. Other strategies in-
clude a pyridine-based strategy and a cyclohexadie-
none photorearrangement approach. Enantioselective
approaches based on alkene metathesis have recently
been reported.
Phenanthroindolizidine alkaloids are pentacyclic natural
products produced by various plants of the Asclepiada-
ceae family, including Cryptocarya and Ficus species.
Biological activities of these compounds include anti-
amoebic, antibacterial, anti-inflammatory, and anti-
fungal effects, as well as significant cytotoxic activity
against various cancer cell lines.1,2 Prototype com-
pounds, such as antofine (1a), not only exhibit IC50 val-
ues in the low nanomolar range, but are also equally
effective against drug-sensitive and multidrug-resistant
(MDR) human cancer cell lines.3,4 Alkaloid 1a exerts
its cytotoxicity by inhibiting protein and nucleic acid
synthesis. Recent mechanism studies by Lee et al. indi-
cated that 1a exhibits inhibitory activity on cell prolifer-
ation by arresting the G2/M phase of the cell cycle.5
Compound 1a also interacts with the bulged regions of
DNA and RNA.6,7
Keywords: Antofine; Deoxypergularinine; Cytotoxic activity; Phenan-
throindolizidine alkaloids.
*
Corresponding author. Tel.: +886 6 2747538; fax: +886 6 2740552;
e-mail: tswu@mail.ncku.edu.tw
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.032

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C.-R. Su et al. / Bioorg. Med. Chem. 16 (2008) 6233–6241
Chuang et al.15 previously published an efficient synthe-
sis of the compound type, preparing both unsubstituted
phenanthroindolizine and the racemic alkaloid tylopho-
rine (2,3,6,7-tetramethoxyphenanthroindolizine). Here-
in, we describe the total synthesis of 1a and 1b, which
are 2,3,6- and 3,6,7-trimethoxy-substituted phenanthro-
indolizidine isomers, respectively, using a similar syn-
thetic protocol. Their dehydroiminium chlorides (2)
and bromides (3) were also prepared. We evaluated in
vitro cytotoxic activity and report structure–activity
relationships (SAR) of these natural alkaloids, their
salts, and certain synthetic intermediates.
bromide to iodide with excess sodium iodide in CH3CN,
radical cyclization with n-Bu3SnH/AIBN in refluxing
toluene)15 to afford phenanthroindolizidinones 14a and
14b. Finally, reduction of 14a and 14b with sodium
bis(2-methoxyethoxy)aluminium hydride in refluxing
dioxane gave racemic antofine 1a and deoxypergulari-
nine 1b, respectively. Dehydroiminium chlorides 2a
and 2b were prepared by exposing CHCl3 solutions of
1a and 1b, respectively, to light and air,17 while the cor-
responding dehydroiminium bromides 3a and 3b were
obtained from oxidation of 1a and 1b by N-bromosuc-
cinimide in CHCl3.18
2. Results and discussion
3. Biological results
Our retrosynthetic analysis of 1a and 1b was identical to
that described and shown in Ref. 15. The starting mate-
rials for our synthesis were the known phenanthryl alde-
hydes 10a and 10b, which were synthesized from the
commercially available acids 4a and 4b and aldehydes
5a and 5b via a conventional five-step sequence, accord-
ing to a previously reported procedure, as shown in
Scheme 1.16
In order to explore their cytotoxic spectra and critical
drug-resistance profiles, the target compounds and
several synthetic intermediates were screened against
four human tumor cell lines, A549 (lung), MCF-7
(breast), KB (nasopharyngeal), and KB-VIN (multi-
drug-resistant KB subline).19 The results are shown
in Table 1. Compound 1a exhibited the best activity
against all four cell lines, with EC50 values ranging
from 0.16 to 4.5 ng/mL. Compound 1b also showed
pronounced activity with EC50 values from 0.011 to
0.043 lg/mL; however, it was less potent (ca. 10- to
70-fold) than 1a. Both compounds showed no signifi-
cant loss in potency against the drug-resistant KV-
VIN cell line as compared with the parental KB cell
line. Compounds 2a, 2b, 3a, and 3b, which have a
dehydroiminium skeleton, showed reduced potency
against all cell lines with EC50 values ranging from
0.2 to 8.26 lg/mL. Interestingly, 14b with an amide
functionality also had moderate potency against all
four cell lines, with EC50 values from 1.15 to
2.14 lg/mL. The remaining tested intermediates 11a–
13a were inactive in this assay.
As shown in Scheme 2, aldehydes 10a and 10b were con-
verted first to carboxylic acids 11a and 11b and then to
acyl azides 12a and 12b through the reported sequence15
of Wittig reaction with (carboethoxymethylene)tri-
phenyl phosphorane, hydrolysis to give the carboxylic
acid, and treatment with oxalyl chloride and sodium
azide to give the azide. These relatively unstable azides
were used directly in the next reaction after purification
by silica gel chromatography. A sequence involving a
thermal Curtius rearrangement, an electrocyclic reac-
tion, and hydrogen shift in boiling o-dichlorobenzene
in the presence of I2, rather than Hg(OAc)2 as in Ref.
15, gave good yields of the isoquinolinones 13a and
13b from 12a and 12b, respectively. The pyrrolidine ring
was added by the established methodology (N-alkyl-
ation with 1-bromo-3-chloropropane, conversion of
Our data confirmed and extended SAR findings
reported previously for this compound class.4,15,20
R1
R1
H3CO
H3CO
H3CO
R1
COOCH3
COOH
CHO
a
b
H3CO
H3CO
COOH
R2
H3CO
R2
R2
7a R1=OCH3, R2=H
7b R1=H, R2=OCH3
6a R1=OCH3, R2=H
6b R1=H, R2=OCH3
4a R1=OCH3
5a R2=H
1
5b R2=OCH3
4b
R =H
R1
R1
R1
H3CO
H3CO
H3CO
O
d
c
COOCH3
e
H
OH
H3CO
H3CO
H3CO
R2
R2
R2
8a R1=OCH3, R2=H
8b R1=H, R2=OCH3
9a R1=OCH3, R2=H
9b R1=H, R2=OCH3
10a R1=OCH3, R2=H
10b R1=H, R2=OCH3
Scheme 1. Reagents and conditions: (a) i—Ac2O, Et3N, reflux, 10 h; ii—K2CO3(aq), 2 h; (b) H2SO4, CH3OH, reflux, 4 h; (c) VOF3, CH2Cl2,
CF3COOH, (CF3CO)2, ice-salt bath, 1 h; (d) LiAlH4, THF, rt, 3 h; (e) PCC, CH2Cl2, rt, 4 h.

C.-R. Su et al. / Bioorg. Med. Chem. 16 (2008) 6233–6241
R1
6235
R1
R1
H3CO
H3CO
H3CO
H3CO
O
O
R3
H
c
a,b
NH
O
H3CO
H3CO
R2
13a R1=OCH3, R2=H
R2
11a R1=OCH3, R2=H, R3=OH
R2
10a R1=OCH3, R2=H
1
2
3
1
2
1
2
11b
12a
12b
R =H, R =OCH3, R =OH
13b
10b
R =H, R =OCH3
R =H, R =OCH3
1
2
3
R =OCH3, R =H, R =N3
1
2
3
R =H, R =OCH3, R =N3
R1
R1
R1
H3CO
H3CO
H3CO
d,e
g
f
N+
X-
N
N
O
1
2
-
H3CO
H3CO
2a
2b
R =OCH3, R =H, X=Cl
H3CO
R2
R2
R2
14a R1=OCH3, R2=H
1
2
-
R =H, R =OCH3, X=Cl
1
2
1a
1b
R =OCH3, R =H
R =H, R =OCH3
3a R1=OCH3, R2=H, X=Br-
1
2
h
1
2
14b
R =H, R =OCH3
1
2
-
3b
R =H, R =OCH3, X=Br
Scheme 2. Reagents and conditions: (a) i—Ph3P@CHCO2Et, toluene, reflux, 4 h; ii—KOH, EtOH–H2O, reflux, 3 h; (b) i—(COCl)2, toluene, 80 ꢁC,
5 h; ii—NaN3, acetone, rt, 2 h; (c) I2, o-dichlorobenzene, reflux, 1 h; (d) i—Br(CH2)3Cl, DMF, rt, overnight; ii—NaI, CH3CN, reflux, overnight; (e)
AIBN, n-Bu3SnH, toluene, reflux, 6 h; (f) NaAl(OCH2CH2OMe)2H2, dioxane, 2 h; (g) light, CHCl3; (h) NBS, CHCl3, rt, 1 h.
For example, Fu et al. found that changing the 2-
methoxy of 1a to a bulkier isopropoxy or more
polar hydroxy group decreased cytotoxic activity.4
In our study, we further found that the absence of
the 2-methoxy as in 1b also decreases potency. Spe-
cifically, decreased cytotoxicity (10- to 70-fold) was
observed for 1b relative to 1a, indicating that the
C-2 methoxy group on the phenanthrene ring is
important for optimal activity.
Table 1. Effect of 1–3, 11a–13a, and 15b against tumor cell line
replicationa
Compound
EC50 (lg/mL)
A549
MCF-7
KB
KB-VIN
1a
1b
0.00016
0.011
0.73
1.06
0.49
0.32
NA
0.0045
0.043
1.72
1.49
1.29
1.18
NA
0.00043
0.023
0.93
0.00083
0.016
0.33
2a
2b
0.95
5.37
5.66
5.14
3a
3b
0.29
NA
8.26
R1
2
R1
11a
12a
13a
14b
14.72
9.36
7.39
NA
NA
8.29
NA
13.52
NA
1.39
H3CO
3
H3CO
1
8
14
9
13
13a
4
1.15
2.14
1.16
12
a Cell line: A549 = lung; MCF-7 = breast; KB = nasopharynx; KB-
VIN = nasopharynx MDR.
N+
X-
N
5
11
6
H3CO
H3CO
7
R2
R2
2a R1=OCH3, R2=H, X=Cl-
1a R1=OCH3, R2=H
Introduction of double bonds adjacent to the phenan-
threne nucleus reduced activity substantially. Com-
pounds 1a/1b (non-planar) showed greater potency
than their respective dehydro-counterparts, 2a/2b
and 3a/3b (planar). The dehydro compounds have a
charged pyridinium ring, which could decrease cellu-
lar permeability. In addition, compounds 1a/1b were
much more potent than 14a/14b. The ketone at posi-
tion-9 in the latter compounds changes the tertiary
amine in the indolizidine nucleus to an amide, and
increases the rigidity and planarity. The five-ring sys-
tem is essential for cytotoxic activity; isoquinoline 13b
(four-ring) and phenanthrenes 11a and 12a (three-
ring) were inactive in all cell lines. Recently, several
groups have reported that cytotoxic potency of phen-
anthroindolizidine is influenced by the substitution
types and patterns on the phenanthrene ring.4,20
1
2
-
1
2
2b
R =H, R =OCH3, X=Cl
1b
R =H, R =OCH3
3a R1=OCH3, R2=H, X=Br-
3b R1=H, R2=OCH3, X=Br-
In summary, total synthesis of two natural phenan-
throindolizidine alkaloids antofine (1a) and deoxyper-
gularinine (1b) and their dehydroiminium chlorides
(2a and 2b) and bromides (3a and 3b) were accom-
plished using a methodology based on thermal and
radical cyclizations and a Curtius rearrangement.
Alkaloids 1a and 1b showed profound cytotoxic activ-
ity against four cancer cell lines, with comparable po-
tency in KB and KB-VIN cell lines. From activity
results, we demonstrated that a non-planar five-ring
structure and a methoxy group at the 2-position are
important and favorable for potent activity of phenan-
throindolizidine alkaloids.

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C.-R. Su et al. / Bioorg. Med. Chem. 16 (2008) 6233–6241
4. Experimental
4.3. Preparation of stilbene methyl esters 7a and 7b
4.1. General methods
4.3.1. (E)-2-(3,4-Dimethoxyphenyl)-3-(4-methoxyphenyl)-
acrylic acid methyl ester 7a. Stilbene acid 6a (6.28 g,
20 mmol) was dissolved in 120 mL water containing
8 mL concentrated H2SO4, and the resulting solution
was refluxed for 4 h. The solvent was evaporated under
reduced pressure, and the crude residue was purified by
silica gel column chromatography (n-hexane–
CHCl3 = 1/2) to afford 7a (4.21 g, 63.1%): yellow crys-
tals, mp 111–112 ꢁC, IR (KBr) cmꢀ1: 3000, 2951, 1708,
Melting points were determined on a Yanaco MP-S3
melting point apparatus and are uncorrected. IR spectra
were determined as KBr discs on a Shimadzu FT-IR
1
Prestige 21. H and 13C NMR spectra were recorded
on Bruker Avance NMR 300 and AV 500 spectrome-
ters, using tetramethylsilane (TMS) as an internal stan-
dard; all chemical shifts were reported in ppm (d). All
MS and HRMS spectra (EI) were obtained on a VG-
70-250S mass spectrometer. FAB and HR-FAB mass
spectra were obtained on a Joel JMS-700 spectrometer.
Tetrahydrofuran was distilled over sodium, and dichlo-
romethane was distilled over CaH2 unless otherwise sta-
ted. All other solvents and chemicals were used as
purchased unless otherwise stated. Reactions were per-
formed under Ar atmosphere in oven-dried flasks unless
otherwise indicated.
1
1602; H NMR (300 MHz, CDCl3) d: 7.77 (1H, s, H-
300), 7.02 (2H, d, J = 8.8 Hz, H-20 and H-60), 6.90 (1H,
d, J = 8.1 Hz, H-5), 6.78 (1H, dd, J = 8.1, 1.2 Hz, H-
6), 6.74 (1H, d, J = 1.2 Hz, H-2), 6.70 (2H, d,
J = 8.8 Hz, H-30 and H-50), 3.92 (3H, s, OCH3-4), 3.80
(3H, s, OCH3-3), 3.79 (3H, s, OCH3-1100), 3.76 (3H, s,
OCH3-40); 13C NMR (75 MHz, CDCl3) d: 168.7,
160.3, 149.1, 148.5, 140.2, 132.4·2, 129.6, 128.6, 127.3,
122.1, 113.7·2, 112.8, 111.4, 55.8, 55.7, 55.2, 52.3; EIMS
m/z: 328 ([M+], 100), 269 (10), 151 (58). Anal. Calcd for
C19H20O5: C, 69.50; H, 6.14. Found: C, 69.63; H, 6.11.
4.2. Preparation of stilbene acids 6a and 6b
4.2.1. (E)-2-(3,4-Dimethoxyphenyl)-3-(4-methoxyphenyl)-
acrylic acid 6a. A solution of homoveratric acid
(2.78 g, 20 mmol), p-anisaldehyde (3.96 g, 20 mmol),
acetic anhydride (5.7 mL), and triethylamine (2.1 mL)
was refluxed with stirring under Ar for 10 h. The result-
ing solution was cooled to room temperature, 10%
K2CO3 solution (100 mL) was added, and then the
resulting mixture was stirred for 2 h at 60 ꢁC. The reac-
tion mixture was cooled to room temperature and neu-
tralized with concentrated HCl (pH 4). The yellow
stilbene acid obtained was collected by filtration and
recrystallized from CH3OH to yield pure6a (4.96 g,
4.3.2.
(E)-3-(3,4-Dimethoxyphenyl)-2-(4-methoxyphenyl)-
acrylic acid methyl ester 7b. Esterification of 6b according
to the above procedure afforded 7b (4.21 g, 63.1%): yellow
crystals, mp 72–73 ꢁC, IR (KBr) cmꢀ1: 3005, 2950,
2838,1700, 1604; 1H NMR (300 MHz, CDCl3) d: 7.76
(1H, s, H-300), 7.16 (2H, d, J = 8.5 Hz, H-2 and H-6),
7.04 (2H, d, J = 8.5 Hz, H-3 and H-5), 6.84 (1H, dd,
J = 8.4, 2.2 Hz, H-60), 6.71 (1H, d, J = 8.4 Hz, H-50), 6.47
(1H, s, H-20), 3.82 (3H, s, OCH3-40), 3.80 (3H, s, OCH3-
30), 3.76 (3H, s, OCH3-1100), 3.44 (3H, s, OCH3-4); 13C
NMR (75 MHz, CDCl3) d: 168.5, 159.0, 149.8, 148.0,
140.2, 131.0·2, 129.5, 128.4, 127.4, 125.2, 114.2·2, 112.3,
110.4, 55.6, 55.1, 55.0, 52.1; EIMS m/z: 328 ([M+], 100),
121 (34). Anal. Calcd for C19H20O5: C, 69.50; H, 6.14.
Found: C, 69.48; H, 6.05.
79%): yellow needles, mp 217–218 ꢁC, IR (KBr) cmꢀ1
:
1
3536, 2959, 1679, 1601; H NMR (300 MHz, CDCl3)
d: 7.87 (1H, s, H-300), 7.06 (2H, d, J = 8.4 Hz, H-20 and
H-60), 6.91 (1H, d, J = 8.0 Hz, H-5), 6.82 (1H, d,
J = 8.0 Hz, H-6), 6.76 (1H, s, H-2), 6.71 (2H, d,
J = 8.4 Hz, H-30 and H-50), 3.93 (3H, s, OCH3-4), 3.81
(3H, s, OCH3-3), 3.93 (3H, s, OCH3-40); 13C NMR
(75 MHz, CDCl3) d: 172.5, 160.6, 149.2, 148.7, 142.1,
132.7, 128.6, 128.1, 127.0, 122.1, 113.8, 113.8, 112.8,
111.5, 55.9, 55.8, 55.2; EIMS m/z: 314 ([M+], 100), 178
(9), 137 (17). Anal. Calcd for C18H18O5: C, 68.78; H,
5.77. Found: C, 68.68; H, 5.73.
4.4. Preparation of phenanthrene esters 8a and 8b
4.4.1. 3,6,7-Trimethoxyphenanthrene-9-carboxylic acid
methyl ester 8a. A solution composed of stilbene ester
7a (6.56 g, 20 mmol) and 1 mL of trifluoroacetic acid
anhydride in 60 mL of dry CH2Cl2 was added dropwise
to a solution of vanadium oxytrifluoride (4.96 g,
40 mmol) in 60 mL CH2Cl2 and 30 mL EtOAc contain-
ing 4 mL of trifluoroacetic acid and 1 mL of trifluoro-
acetic anhydride. During the addition (60 min), the
reaction mixture was cooled in an ice-salt bath. The
dark brown mixture was stirred for an additional 0.5 h
in an ice-salt bath, then poured onto crushed ice, and
was extracted with CH2Cl2. The organic layer was
washed with brine, dried over Na2SO4, and concen-
trated under reduced pressure. The residue was purified
by silica gel column chromatography (n-hexane–
CHCl3 = 1/4) to afford 8a (4.82 g, 73.9%): colorless crys-
tals, mp 150–151 ꢁC, IR (KBr) cmꢀ1: 3116, 2943, 1713,
4.2.2. (E)-3-(3,4-Dimethoxyphenyl)-2-(4-methoxypheny-
l)acrylic acid 6b. Homoanisic acid 4b and 3,4-dimethoxy-
benzaldehyde 5b were used as starting materials in an
analogous procedure as for the preparation of 6a to give
6b (4.75 g, 76%): yellow needles, mp 218–219 ꢁC, IR
:
(KBr) cmꢀ1 3536, 2983, 1663, 1595; 1H NMR
(300 MHz, CDCl3) d: 7.86 (1H, s, H-300), 7.20 (2H, d,
J = 8.5 Hz, H-2 and H-6), 6.95 (2H, d, J = 8.0 Hz, H-3
and H-5), 6.81 (1H, dd, J = 8.3, 1.5 Hz, H-60), 6.73
(1H, d, J = 8.3 Hz, H-50), 6.53 (1H, d, J = 1.5 Hz, H-
20), 3.84 (3H, s, OCH3-40), 3.81 (3H, s, OCH3-30), 3.46
(3H, s, OCH3-4); 13C NMR (75 MHz, CDCl3) d: 173.1,
159.3, 150.3, 148.2, 142.2, 131.2·2, 128.0, 127.3, 125.8,
114.3·2, 112.5, 110.5, 55.8, 55.3, 55.2; EIMS m/z: 314
([M+], 100), 178 (9), 137 (17). Anal. Calcd for
C18H18O5: C, 68.78; H, 5.77. Found: C, 68.58; H, 5.77.
1
1613; H NMR (300 MHz, CDCl3) d: 8.64 (1H, s, H-
8), 8.42 (1H, s, H-10), 7.84 (1H, d, J = 8.6 Hz, H-1),
7.83 (1H, s, H-5), 7.78 (1H, d, J = 2.4 Hz, H-4), 7.20
(1H, dd, J = 8.6, 2.4 Hz, H-2), 4.10 (3H, s, OCH3-6),
4.08 (3H, s, OCH3-7), 4.02 (3H, s, OCH3-3), 4.00 (3H,

C.-R. Su et al. / Bioorg. Med. Chem. 16 (2008) 6233–6241
6237
s, OCH3-11); 13C NMR (75 MHz, CDCl3) d: 168.2,
160.1, 149.8, 148.8, 133.3, 131.8, 131.2, 125.1, 124.9,
124.2, 121.6, 116.0, 106.9, 103.6, 103.1, 55.8·2, 55.5,
52.0; EIMS m/z: 326 ([M+], 100), 283 (14). Anal. Calcd
for C19H18O5: C, 69.93; H, 5.56. Found: C, 69.70; H,
5.53.
Calcd for C18H18O4: C, 72.47; H, 6.08. Found: C,
72.43; H, 6.06.
4.6. Preparation of phenanthrene aldehydes 10a and 10b
4.6.1. 3,6,7-Trimethoxyphenanthren-9-carbaldehyde 10a.
To a solution of 9a (5.96 g, 20 mmol) in 150 mL CH2Cl2
was added pyridinium chlorochromate (5.17 g,
20 mmol), and the reaction mixture was stirred under
Ar at room temperature for 2.5 h. Aqueous 100 mL
NaHCO3 was added, the mixture was filtered on Celite,
and the filtrate was extracted with CH2Cl2. The organic
layer was washed with brine, dried over Na2SO4, and
concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (CHCl3)
to afford 10a [4.98 g (84.1%)]: colorless crystals, mp
169–170 ꢁC, IR (KBr) cmꢀ1: 2997, 2936, 1672, 1615,
1518; 1H NMR (300 MHz, CDCl3) d: 10.06 (1H, s,
CHO-9), 8.77 (1H, s, H-8), 7.80 (1H, s, H-10), 7.73
(1H, d, J = 8.7 Hz, H-1), 7.55 (2H, s, H-4 and H-5),
7.13 (1H, dd, J = 8.7, 1.6 Hz, H-2), 4.03 (3H, s,
OCH3-6), 4.00 (3H, s, OCH3-7), 3.97 (3H, s, OCH3-3);
13C NMR (75 MHz, CDCl3) d: 193.6, 160.9, 150.3,
148.9, 140.0, 133.9, 131.9, 127.2, 124.5, 124.0, 123.6,
115.9, 106.0, 103.7, 102.7, 55.8, 55.6, 55.4; EIMS m/z:
296 ([M+], 100), 253 (20). Anal. Calcd for C18H16O4:
C, 72.96; H, 5.44. Found: C, 72.88; H, 5.38.
4.4.2. 2,3,6-Trimethoxyphenanthrene-9-carboxylic acid
methyl ester 8b. Phenanthrene ester 8b (4.73 g, 72.5%)
was prepared from 7b according to the above procedure:
colorless crystals, mp 154–155 ꢁC, IR (KBr) cmꢀ1: 3001,
1
2950, 1708, 1617; H NMR (300 MHz, CDCl3) d: 8.91
(1H, d, J = 9.2 Hz, H-8), 8.23 (1H, s, H-10), 7.79 (1H,
s, H-5), 7.74 (1H, s, H-4), 7.24 (1H, d, J = 9.2 Hz, H-
7), 7.18 (1H, s, H-1), 4.08 (3H, s, OCH3-6), 4.00 (9H,
s, OCH3-2,3, and 6); 13C NMR (75 MHz, CDCl3) d:
168.1, 157.9, 159.8, 149.5, 131.5, 129.4, 128.2, 126.6,
125.4, 123.6, 123.1, 115.6, 109.1, 104.2, 103.0, 55.9,
55.8, 55.4, 52.0; EIMS m/z: 326 ([M+], 100), 181 (20).
Anal. Calcd for C19H18O5: C, 69.93; H, 5.56. Found:
C, 69.61; H, 5.52.
4.5. Preparation of phenanthrene alcohols 9a and 9b
4.5.1. (3,6,7-Trimethoxyphenanthrene-9-yl)methanol 9a.
To a suspension of LiAlH4 (2.4 g, 20 mmol) in 70 mL of
dry THF was added a solution of 8a (6.52 g, 20 mmol)
in 100 mL of dry THF. The reaction mixture was stirred
under Ar for 3 h and quenched by sequential addition of
4 mL of water, 4 mL of 15% NaOH, and 8 mL of water.
The mixture was filtered, and the filtrate was concen-
trated under reduced pressure. The residue was dis-
solved in CHCl3, and the solution was washed with
brine, dried over Na2SO4, and concentrated under re-
duced pressure. The residue was purified by silica gel
column chromatography (CHCl3) to afford 9a (4.96 g,
83.2%): colorless crystals, mp 155–156 ꢁC, IR (KBr)
cmꢀ1: 3386, 2933, 1610, 1525; 1H NMR (300 MHz,
CDCl3) d: 7.83 (1H, s, H-5), 7.77 (1H, d, J = 1.6 Hz,
H-4), 7.73 (1H, d, J = 8.6 Hz, H-1), 7.55 (1H, s, H-10),
7.49 (1H, s, H-8), 7.17 (1H, dd, J = 8.6, 1.6 Hz, H-2),
5.05 (2H, d, J = 4.1 Hz, H-11), 4.08 (3H, s, OCH3-6),
4.03 (3H, s, OCH3-7), 4.00 (3H, s, OCH3-3), 1.87 (1H,
br s, D2O-exchangeable, OH-11); 13C NMR (75 MHz,
CDCl3) d: 158.3, 149.4, 148.8, 131.3, 131.2, 130.2,
125.7, 125.5, 124.8, 124.5, 115.4, 104.8, 103.9, 103.7,
64.7, 55.9·2, 55.5; EIMS m/z: 298 ([M+], 100), 152
(21). Anal. Calcd for C18H18O4: C, 72.47; H, 6.08.
Found: C, 72.37; H, 6.06.
4.6.2. 2,3,6,-Trimethoxyphenanthren-9-carbaldehyde 10b.
Phenanthrene aldehyde 10b (4.89 g, 82.6%) was pre-
pared according to the above procedure: colorless crys-
tals, mp 183–184 ꢁC, IR (KBr) cmꢀ1: 3011, 2960, 2938,
1679, 1616, 1515; 1H NMR (300 MHz, CDCl3) d:
10.25 (1H, s, CHO-9), 9.31 (1H, d, J = 9.2 Hz, H-8),
7.97 (1H, s, H-10), 7.83 (1H, d, J = 2.2 Hz, H-5), 7.80
(1H, s, H-4), 7.30 (1H, dd, J = 9.2, 2.2 Hz, H-7), 7.29
(1H, s, H-1), 4.12 (3H, s, OCH3-6), 4.05 (3H, s,
OCH3-3), 4.02 (3H, s, OCH3-2); 13C NMR (75 MHz,
CDCl3) d: 193.5, 158.6, 151.9, 149.8, 138.3, 131.7,
128.8, 127.7·2, 125.6, 122.2, 116.1, 109.5, 104.6, 103.3,
56.1·2, 55.5; EIMS m/z: 296 ([M+], 100), 281 (6). Anal.
Calcd for C18H16O4: C, 72.96; H, 5.44. Found: C, 72.99;
H, 5.42.
4.7. Preparation of phenanthrene acrylic acids 11a and
11b
4.7.1. (E)-3-(3,6,7-Trimethoxyphenanthren-9-yl)acrylic
acid 11a. A mixture of 10a (2.96 g, 10 mmol) and (carbo-
ethoxymethylene)triphenylphosphorane
(4.76 g,
4.5.2. (2,3,6-Trimethoxyphenanthrene-9-yl)methanol 9b.
Phenanthrene alcohol9b (4.97 g, 83.5%) was obtained
using 8b according to the above procedure: colorless
crystals, mp 185–186 ꢁC, IR (KBr) cmꢀ1: 3217, 2951,
13 mmol) in 80 mL of toluene was refluxed under Ar
for 5 h. After cooling, the resulting solution was directly
purified by silica gel column chromatography (n-hex-
ane–CHCl3 = 1/4) and afforded the ethyl ester (3.19 g,
87.2%). Then, a solution of 30 mL 1 N KOH was added
to a solution of the ethyl ester (3.19 g, 8.7 mmol) in
60 mL EtOH, and the reaction mixture was heated un-
der reflux for 3 h. After cooling, the solvent was evapo-
rated under reduced pressure. The residue was dissolved
in 30 mL water and acidified with 10% HCl. The yellow
solid was filtered and recrystallized from CH3OH to af-
ford pure11a (2.68 g, 91.2%): yellow crystals, mp 258–
259 ꢁC, IR (KBr) cmꢀ1: 3530, 2946, 1679, 1605, 1519;
1
1611, 1512; H NMR (300 MHz, CDCl3) d: 8.10 (1H,
d, J = 9.0 Hz, H-9), 7.88 (1H, d, J = 2.2 Hz, H-5), 7.85
(1H, s, H-4), 7.55 (1H, s, H-10), 7.25 (1H, dd, J = 9.0,
2.2 Hz, H-7), 7.19 (1H, s, H-1), 5.13 (2H, s, H-11),
4.10 (3H, s, OCH3-6), 4.03 (6H, s, OCH3-2 and 3),
4.00 (3H, s, OCH3-3); 13C NMR (75 MHz, CDCl3) d:
158.1, 149.6, 149.4, 132.8, 131.7, 127.0, 126.1, 124.4,
124.1, 123.2, 115.2, 108.4, 104.7, 103.4, 64.3, 55.1,
55.9, 55.5; EIMS m/z: 298 ([M+], 100), 283 (6). Anal.

6238
C.-R. Su et al. / Bioorg. Med. Chem. 16 (2008) 6233–6241
1H NMR (300 MHz, CDCl3) d: 12.50 (1H, br s, D2O-
exchangeable, OH), 8.34 (1H, d, J = 15.7 Hz, H-11),
8.12 (1H, s, H-8), 8.10 (1H, s, H-10), 8.06 (1H, s, H-
4), 7.92 (1H, d, J = 8.8 Hz, H-1), 7.47 (1H, s, H-5),
7.24 (1H, d, J = 8.8 Hz, H-2), 6.61 (1H, d, J = 15.7 Hz,
H-12), 4.04 (3H, s, OCH3-7), 4.00 (3H, s, OCH3-6),
3.97 (3H, s, OCH3-3); 13C NMR (75 MHz, CDCl3) d:
167.5, 158.9, 149.5, 149.0, 141.4, 131.6, 130.9, 126.7,
124.9·2, 124.5, 124.1, 120.9, 116.5, 104.7, 104.1·2,
55.9, 55.6, 55.4; EIMS m/z: 338 ([M+], 100), 295 (37),
277 (62). Anal. Calcd for C20H18O5: C, 70.99; H, 5.36.
Found: C, 70.82; H, 5.35.
crystals, mp 130–131 ꢁC, IR (KBr) cmꢀ1: 3008, 2938,
2834, 2141, 1678, 1613, 1513; 1H NMR (300 MHz,
CDCl3) d: 8.51 (1H, d, J = 15.5 Hz, H-11), 8.07 (1H,
d, J = 9.0 Hz, H-8), 7.83 (1H, s, H-5), 7.75 (1H, s, H-
10), 7.24 (1H, d, J = 9.0 Hz, H-7), 7.16 (1H, s, H-1),
6.53 (1H, d, J = 15.5 Hz, H-12), 4.10 (3H, s, OCH3-6),
4.03 (3H, s, OCH3-3), 4.02 (3H, s, OCH3-2); 13C
NMR (75 MHz, CDCl3) d: 172.0, 158.3, 150.3, 149.7,
144.4, 131.3, 127.8, 126.5, 125.8, 125.5, 124.1, 123.8,
120.5, 115.6, 108.7, 104.6, 103.1, 56.0, 55.9, 55.5; EIMS
m/z: 335 ([M+ꢀN2], 100), 320 (11). Anal. Calcd for
C20H17N3O4: C, 66.11; H, 4.72; N, 11.56. Found: C,
66.46; H, 4.82; N, 11.08.
4.7.2. (E)-3-(2,3,6-Trimethoxyphenanthren-9-yl)acrylic
acid 11b. Phenanthrene acrylic acid 11b (2.75 g, 92.5%)
was prepared according to the above procedure: yellow
crystals, mp 205–206 ꢁC, IR (KBr) cmꢀ1: 3530, 2953,
4.9. Preparation of isoquinolinones 13a and 13b
4.9.1.
6,7,10-Trimethoxy-2H-2-azatriphenylene-1-one
1
2937, 1689, 1616, 1512; H NMR (300 MHz, CDCl3)
13a. A mixture of azide 12a (1.1 g, 3 mmol) and I2
(0.02 g, catalytic amount) in 30 mL o-dichlorobenzene
was refluxed for 2 h. After cooling, compound 13a was
isolated by filtration and washed with n-hexane. The
crude product was recrystallized from a CH3OH–CHCl3
mixture to afford pure isoquinolinone 13a (839 mg,
83.5%): pale yellow crystals, mp 269–270 ꢁC, IR (KBr)
cmꢀ1: 3272, 2939, 2890, 1630, 1614, 1555, 1503; 1H
NMR (300 MHz, DMSO-d6) d: 11.70 (1H, br s, D2O-
exchangeable, NH), 10.30 (1H, d, J = 9.5 Hz, H-12),
8.10 (2H, s, H-8 and H-9), 7.93 (1H, s, H-5), 7.47 (2H,
m, H-3 and H-4), 7.27 (1H, d, J = 9.5 Hz, H-11), 4.07
(3H, s, OCH3-7), 4.01 (3H, s, OCH3-6), 3.99 (3H, s,
OCH3-10); 13C NMR (75 MHz, DMSO-d6) d: 162.6,
157.7, 150.9, 149.6, 136.3, 130.3, 130.1, 129.0, 126.2,
123.2, 122.1, 117.0, 115.0, 106.1, 105.0, 104.7, 101.0,
55.9, 55.8, 55.3; EIMS m/z: 335 ([M+], 100), 292 (17).
Anal. Calcd for C20H17NO4: C, 71.63; H, 5.11; N,
4.18. Found: C, 71.22; H, 5.17; N, 4.03.
d: 12.40 (1H, br s, D2O-exchangeable, OH), 8.34 (1H,
d, J = 15.6 Hz, H-11), 8.12 (1H, d, J = 2.0 Hz, H-5),
8.10 (1H, d, J = 9.2 Hz, H-8), 8.09 (1H, s, H-10), 8.06
(1H, s, H-4), 7.49 (1H, s, H-4), 7.29 (1H, dd, J = 9.2,
2.0 Hz, H-7), 6.57 (1H, d, J = 15.6, H-12), 4.04 (3H, s,
OCH3-6), 4.00 (3H, s, OCH3-3), 3.92 (3H, s, OCH3-2);
13C NMR (75 MHz, CDCl3) d: 169.5, 158.0, 150.0,
149.6, 141.2, 131.1, 127.5, 126.4, 125.3, 125.0, 123.4·2,
120.8, 116.1, 109.1, 104.9, 104.1, 56.0, 55.6, 55.5; EIMS
m/z: 338 ([M+], 100), 293 (8). Anal. Calcd for C20H18O5:
C, 70.99; H, 5.36. Found: C, 70.95; H, 5.34.
4.8. Preparation of phenanthrene acryloyl azides 12a and
12b
4.8.1. (E)-3-(3,6,7-Trimethoxyphenanthren-9-yl)acryloyl
azide 12a. A mixture of 11a (1.69 g, 5 mmol) and oxalyl
chloride (1.27 g, 10 mmol) in 70 mL of toluene was
heated for 5 h at 80 ꢁC. After cooling, the resulting mix-
ture was concentrated under reduced pressure to afford
the acyl chloride quantitatively. The acyl chloride was
added immediately to a suspension of NaN3 (0.98 g,
15 mmol) in 60 mL dry acetone in an ice bath. The reac-
tion mixture was stirred for 2 h at room temperature
and filtered. The solvent was evaporated under reduced
pressure, and the residue was purified by silica gel col-
umn chromatography (CHCl3) to afford 12a (1.67 g,
92.1%): yellow crystals, mp 121–122 ꢁC, IR (KBr)
4.9.2. 7,10,11-Trimethoxy-2H-2-azatriphenylene-1-one
13b. Isoquinolinone 13b (820 mg, 81.6%) was prepared
according to the above procedure: pale yellow crystals,
mp 274–275 ꢁC, IR (KBr) cmꢀ1: 3267, 3219, 2957,
1
1627, 1554, 1517; H NMR (300 MHz, DMSO-d6) d:
11.70 (1H, br s, D2O-exchangeable, NH), 10.04 (1H, s,
H-12), 8.56 (1H, d, J = 9.2 Hz, H-5), 8.11 (1H, d,
J = 2.0 Hz, H-8), 8.10 (1H, s, H-9), 7.44 (1H, m, H-4),
7.43 (1H, m, H-3), 7.29 (1H, dd, J = 9.2, 2.0 Hz, H-6),
4.04 (3H, s, OCH3-11), 4.03 (3H, s, OCH3-10), 3.92
(3H, s, OCH3-7); 13C NMR (75 MHz, DMSO-d6) d:
162.7, 160.1, 149.0, 148.4, 137.3, 133.2, 130.4, 127.3,
125.0, 123.3, 120.6, 116.2, 115.7, 108.7, 105.1, 104.5,
100.6, 55.7, 55.6, 55.2; EIMS m/z: 335 ([M+], 100), 320
(12), 293 (17). Anal. Calcd for C20H17NO4: C, 71.63;
H, 5.11; N, 4.18. Found: C, 71.78; H, 5.13; N, 4.22.
cmꢀ1
:
2933, 2140, 1674, 1604, 1509; 1H NMR
(300 MHz, CDCl3) d: 8.45 (1H, d, J = 15.5 Hz, H-11),
7.84 (2H, s, H-8 and H-10), 7.78 (1H, d, J = 8.6 Hz,
H-1), 7.76 (1H, d, J = 1.6 Hz, H-4), 7.38 (1H, s, H-5),
7.20 (1H, dd, J = 8.6, 1.6 Hz, H-2), 6.54 (1H, d,
J = 15.5, H-12), 4.10 (3H, s, OCH3-7), 4.07 (3H, s,
OCH3-7), 3.92 (3H, s, OCH3-3); 13C NMR (75 MHz,
CDCl3) d: 172.0, 159.4, 149.8, 149.2, 144.5, 132.4,
131.2, 126.6, 125.5, 125.4, 125.1, 124.5, 120.4, 116.1,
104.0, 103.8, 103.7, 56.0·2, 55.5; EIMS m/z: 335
([M+ꢀN2], 100). Anal. Calcd for C20H17N3O4: C,
66.11; H, 4.72; N, 11.56. Found: C, 66.82; H, 4.80; N,
11.07.
4.10. Preparation of phenanthroindolizidiones 14a and
14b
4.10.1. 2-(3-Chloropropyl)-6,7,10-trimethoxy-2H-2-azat-
riphenylen-1-one. To a suspension of NaH (60% disper-
sion in oil, 80 mg, 2 mmol) in DMF (5 mL) cooled in an
ice bath, a solution of 13a (335 mg, 1 mmol) in DMF
(15 mL) was added with stirring. After the addition
was completed, the mixture was added dropwise to a
4.8.2. (E)-3-(2,3,6-Trimethoxyphenanthren-9-yl)acryloyl
azide 12b. Phenanthrene acryloyl azide 12b (1.64 g,
90.3%) was obtained using the above procedure: yellow

C.-R. Su et al. / Bioorg. Med. Chem. 16 (2008) 6233–6241
6239
solution of 1-bromo-3-chloropropane (628 mg, 4 mmol)
in 5 mL DMF. The mixture was stirred at room temper-
ature overnight. The solvent was evaporated in vacuo,
and water was then added. The mixture was extracted
with CHCl3, washed with brine, dried over Na2SO4,
and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (n-
hexane–CHCl3 = 1/4) to afford the chloropropyl iso-
quinolinone of 13a (292 mg, 71.2%): colorless crystals,
mp 183–184 ꢁC, IR (KBr) cmꢀ1: 2988, 2934, 1644,
(KBr) cmꢀ1: 3119, 2957, 1644, 1614, 1599, 1519; 1H
NMR (300 MHz, CDCl3) d: 10.05 (1H, s, H-12), 8.37
(1H, d, J = 9.2 Hz, H-5), 7.91 (1H, s, H-9), 7.90 (1H,
d, J = 2.6 Hz, H-8), 7.47 (1H, d, J = 7.4 Hz, H-3), 7.29
(1H, d, J = 7.4 Hz, H-4), 7.24 (1H, dd, J = 9.2, 2.6 Hz,
H-6), 4.31 (2H, t, J = 6.2 Hz, H-10), 4.15 (3H, s,
OCH3-11), 4.12 (3H, s, OCH3-10), 4.04 (3H, s, OCH3-
7), 3.63 (2H, t, J = 6.2 Hz, H-30), 2.40 (2H, p,
J = 6.2 Hz, H-20); 13C NMR (75 MHz, CDCl3) d:
162.6, 160.3, 149.6, 148.6, 136.8, 133.7, 133.2, 126.6,
125.6, 124.1, 120.9, 117.2, 115.1, 109.1, 104.9, 103.4,
101.5, 56.0,55.8, 55.5, 47.7, 42.1, 31.2; EIMS m/z: 413
([M+2], 37), 411 ([M+], 100), 376 (28). Anal. Calcd for
C23H22ClNO4: C, 67.07; H, 5.38; N, 3.40. Found: C,
67.07; H, 5.36; N, 3.37.
1
1598, 1528; H NMR (300 MHz, CDCl3) d: 10.31 (1H,
d, J = 9.5 Hz, H-12), 7.93 (2H, s, H-8 and H-9), 7.73
(1H, s, H-5), 7.48 (1H, d, J = 7.3 Hz, H-3), 7.32 (1H,
d, J = 9.5 Hz, H-11), 7.21 (1H, d, J = 7.3 Hz, H-4),
4.32 (2H, t, J = 6.4 Hz, H-10), 4.14 (3H, s, OCH3-7),
4.11 (3H, s, OCH3-6), 4.03 (3H, s, OCH3-10), 3.64
(2H, t, J = 5.9 Hz, H-30), 2.40 (2H, tt, J = 6.4, 5.9 Hz,
H-20); 13C NMR (75 MHz, CDCl3) d: 162.1, 158.2,
151.0, 149.6, 135.6, 133.0, 131.1, 130.0, 127.0, 123.8,
122.3, 118.3, 114.5, 105.4, 105.2, 104.0, 101.4, 56.0·2,
55.5, 47.9, 42.2, 31.1; EIMS m/z: 413 ([M+2], 42), 411
([M+], 80), 376 (46). Anal. Calcd for C23H22ClNO4: C,
67.07; H, 5.38; N, 3.40. Found: C, 67.79; H, 5.40; N,
3.39.
4.10.4. 3,6,7-Trimethoxy-11,12,12a,13-tetrahydro-10H-
9a-azacyclopenta[b]triphenylen-9-one 14b. Phenanthro-
indolizidinone 14b (143 mg, 75.8%) was obtained using
the above procedure: colorless crystals, mp 200–
201 ꢁC, IR (KBr) cmꢀ1: 2959, 2937, 1638, 1615, 1516;
1H NMR (300 MHz, CDCl3) d: 9.02 (1H, s, H-8), 8.00
(1H, d, J = 9.0 Hz, H-1), 7.88 (1H, d, J = 2.2 Hz, H-4),
7.86 (1H, s, H-5), 7.22 (1H, dd, J = 9.0, 2.2 Hz, H-2),
4.11 (3H, s, OCH3-7), 4.09 (3H, s, OCH3-6), 4.04 (3H,
s, OCH3-3), 3.87 (1H, m, H-11), 3.83 (1H, m, H-13),
3.79 (1H, m, H-11), 3.66 (1H, dd, J = 15.6, 4.2 Hz, H-
14), 2.93 (1H, dd, J = 15.6, 13.6 Hz, H-14), 2.42 (1H,
m, H-13), 2.15 (1H, m, H-12), 1.93 (1H, m, H-12),
1.90 (1H, m, H-13); 13C NMR (75 MHz, CDCl3)
d:164.6, 159.3, 149.6, 148.6, 134.2, 133.1, 126.7, 125.3,
124.3, 123.0, 122.0, 115.5, 108.1, 104.4, 102.9, 55.9·2,
55.5, 55.3, 45.4, 33.9, 32.3, 23.5; EIMS m/z: 377 ([M+],
100), 308 (38), 280 (28). Anal. Calcd for C23H23NO4:
C, 73.19; H, 6.14; N, 3.71. Found: C, 73.42; H, 6.17;
N, 3.86.
4.10.2. 2,3,6-Trimethoxy-11,12,12a,13-tetrahydro-10H-
9a-azacyclopenta[b]triphenylen-9-one 14a. A solution of
chloropropyl isoquinolinone (251 mg, 0.5 mmol) and
NaI (375 mg, 2.5 mmol) in dry CH3CN (15 mL) was
placed in a sealed tube and then heated at 125 ꢁC for
12 h. After cooling, the reaction mixture was filtered,
and concentrated under reduced pressure to yield the
iodopropyl isoquinolinone quantitatively. Subsequently,
a solution of AIBN (33 mg, 0.2 mmol) in 2.4 mL toluene
was added dropwise (syringe pump) to a degassed solu-
tion of iodopropyl isoquinolinone (251 mg, 0.5 mmol)
and n-Bu3SnH (175 mg, 0.6 mmol) in 25 mL of toluene
at refluxing temperature for 6 h. The reaction mixture
was then cooled and the solvent was removed under re-
duced pressure. The residue was washed with n-hexane,
purified by silica gel column chromatography (CHCl3)
to give phenanthroindolizidione 14a (140 mg, 74.6%):
4.11. Preparation of phenanthroindolizidines 1a and 1b
4.11.1. Antofine 1a. To a solution of 15a (39.7 mg,
1 mmol) in dry 5 mL dioxane was added a 3.5 M solu-
tion of sodium bis(2-methoxyethoxy)aluminium hydride
in toluene (0.4 mL, 1.4 mmol) and the mixture was re-
fluxed for 2 h in the dark. After evaporation of the sol-
vents, the residue was diluted with 10 mL H2O and then
basified with 10 mL 10% aqueous NaOH. The mixture
was extracted with CHCl3, dried over Na2SO4, and con-
centrated under reduced pressure. The residue was puri-
fied by silica gel column chromatography (CHCl3–
MeOH = 19/1) to yield 1a (32.8 mg, 90.3%): colorless
crystals, mp 211–212 ꢁC, IR (KBr) cmꢀ1: 2914, 1615,
colorless crystals, mp 252–253 ꢁC, IR (KBr) cmꢀ1
:
1
2947, 1631, 1614, 1518; H NMR (300 MHz, CDCl3)
d: 9.27 (1H, d, J = 9.3 Hz, H-8), 7.82 (2H, s, H-4 and
H-5), 7.81 (1H, d, J = 9.3 Hz, H-7), 7.22 (1H, s, H-1),
4.09 (3H, s, OCH3-3), 4.01 (3H, s, OCH3-2), 3.99 (3H,
s, OCH3-6), 3.86 (1H, m, H-11), 3.83 (1H, m, H-13),
3.80 (1H, m, H-11), 3.46 (1H, dd, J = 15.6, 3.9 Hz, H-
14), 2.80 (1H, dd, J = 15.6, 12.0 Hz, H-14), 2.39 (1H,
m, H-13), 2.12 (1H, m, H-12), 1.89 (1H, m, H-12),
1.82 (1H, m, H-13); 13C NMR (75 MHz, CDCl3) d:
164.3, 157.6, 149.9, 149.4, 132.5, 130.8, 129.5·2, 126.3,
124.2, 123.4, 115.1, 104.7, 104.0, 103.7, 55.9, 55.8,
55.4, 55.1, 45.2, 33.8, 32.5, 23.5; EIMS m/z: 377 ([M+],
100), 308 (57), 280 (45). Anal. Calcd for C23H23NO4:
C, 73.19; H, 6.14; N, 3.71. Found: C, 73.09; H, 6.40;
N, 3.59.
1
1529, 1512; H NMR (300 MHz, CDCl3) d: 7.90 (1H,
s, H-4), 7.89 (1H, d, J = 2.1 Hz, H-5), 7.79 (1H, d,
J = 9.0 Hz, H-8), 7.28 (1H, s, H-1), 7.19 (1H, dd,
J = 9.0, 2.1 Hz, H-7), 4.66 (1H, d, J = 14.9 Hz, H-9),
4.09 (3H, s, OCH3-3), 4.05 (3H, s, OCH3-2), 4.00 (3H,
s, OCH3-6), 3.66 (1H, d, J = 14.9 Hz, H-9), 3.45 (1H,
td, J = 8.8, 2.0 Hz, H-11), 3.30 (1H, dd, J = 15.7,
2.3 Hz, H-14), 2.87 (1H, dd, J = 15.7, 10.5 Hz, H-14),
2.44 (1H, m, H-13a), 2.43 (1H, q, J = 8.8 Hz, H-11),
2.19 (1H, m, H-13), 1.99 (1H, m, H-12), 1.88 (1H, m,
H-12), 1.72 (1H, m, H-13); 13C NMR (100 MHz) d:
157.4 (C-8), 149.3 (C-3), 148.3 (C-2), 130.1 (C-4b),
4.10.3. 2-(3-Chloropropyl)-7,10,11-trimethoxy-2H-2-azat-
riphenylen-1-one. The chloropropyl isoquinolinone of
13b (297 mg, 72.2%) was prepared according to the
above procedure: colorless crystals, mp 184–185 ꢁC, IR

6240
C.-R. Su et al. / Bioorg. Med. Chem. 16 (2008) 6233–6241
127.0 (C-14b), 126.6 (C-8b), 125.5 (C-14a), 124.2 (C-8),
124.1 (C-8a), 123.5 (C-4a), 114.8 (C-7), 104.6 (C-5),
103.9 (C-1), 103.8 (C-4), 60.2 (C-13a), 56.0 (OCH3-3),
55.8 (OCH3-2), 55.5 (OCH3-6), 55.0 (C-11), 53.8 (C-9),
33.6 (C-14), 31.2 (C-13), 21.5 (C-12); EIMS m/z: 363
([M+], 30), 294 (100). Anal. Calcd for C23H25NO3: C,
76.01; H, 6.93; N, 3.85. Found: C, 75.95; H, 6.80; N,
3.98.
3.89 (3H, s, OCH3-7), 3.86 (3H, s, OCH3-6), 3.80 (3H,
s, OCH3-3), 3.41 (2H, t, J = 7.2 Hz, H-13), 2.54 (2H,
p, J = 7.2 Hz, H-12); 13C NMR (75 MHz) d: 162.5,
151.1, 150.9, 150.8, 140.0, 137.7, 134.6, 128.0, 124.3,
124.1, 120.2, 118.5, 116.7, 116.0, 106.1, 104.1·2, 58.6,
57.3, 56.4, 56.3, 31.4, 22.2; EIMS m/z: 360 ([M+ꢀCl],
43), 154 (100). Anal. Calcd for C23H22ClNO3: C,
69.78; H, 5.60; N, 3.54. Found: C, 69.68; H, 5.55; N,
3.48.
4.11.2. Deoxypergularinine 1b. Deoxypergularinine 1b
(33.4 mg, 92.0%) was prepared according to the above
procedure: colorless crystals, 209–210 ꢁC, IR (KBr)
cmꢀ1: 2959, 2912, 1612, 1512; 1H NMR (300 MHz,
CDCl3) d: 7.91 (1H, d, J = 9.2 Hz, H-1), 7.88 (1H, s,
H-5), 7.86 (1H, d, J = 2.5 Hz, H-4), 7.21 (1H, dd,
J = 9.2, 2.5 Hz, H-2), 7.09 (1H, s, H-8), 4.62 (1H, d,
J = 14.5 Hz, H-9), 4.09 (3H, s, OCH3-7), 4.03 (3H, s,
OCH3-6), 4.01 (3H, s, OCH3-3), 3.72 (1H, d,
J = 14.5 Hz, H-9), 3.47 (1H, td, J = 8.1, 2.0 Hz, H-11),
3.40 (1H, dd, J = 16.5, 3.0 Hz, H-14), 3.00 (1H, dd,
J = 16.5, 10.5 Hz, H-14), 2.59 (1H, m, H-13a), 2.58
(1H, dd, J = 8.1, 8.1 Hz, H-11), 2.25 (1H, m, H-13),
2.06 (1H, m, H-12), 1.96 (1H, m, H-12), 1.82 (1H, m,
H-13); 13C NMR (100 MHz) d: 157.7 (C-3), 149.4 (C-
7), 148.3 (C-6), 130.4 (C-4b), 126.6 (C-8b), 125.3 (C-
14b), 125.2 (C-14a), 125.1 (C-1), 124.2 (C-8a), 123.4
(C-4a), 114.9 (C-2), 104.6 (C-4), 103.8 (C-5), 102.9 (C-
8), 60.2 (C-13a), 56.0 (OCH3-7), 55.9 (OCH3-6), 55.5
(OCH3-3), 55.4 (C-11), 53.1 (C-9), 32.6 (C-14), 30.9
(C-13), 21.5 (C-12); EIMS m/z: 363 ([M+], 40), 294
(100). Anal. Calcd for C23H25NO3: C, 76.01; H, 6.93;
N, 3.85. Found: C, 75.85; H, 6.81; N, 3.97.
4.13. Preparation of dehydrophenanthroindolizidine bro-
mides 3a and 3b
4.13.1. Dehydroantofine bromide 3a. To a solution of
antofine 1a (36.3 mg, 1 mmol) in 10 mL CHCl3 was
added NBS (150 mg, 0.4 mmol) in small portions with
stirring. After stirring for 1 h at room temperature, the
orange solid formed was collected by filtration. The
crude product was purified by aluminum oxide gel chro-
matography (CHCl3–MeOH = 9/1) to provide 3a
(23.1 mg, 52.6%): yellow crystals, >290 ꢁC, IR (KBr)
cmꢀ1: 2970, 1637, 1610, 1521; 1H NMR (300 MHz,
CD3OD) d: 9.91 (1H, s, H-9), 8.76 (1H, s, H-14), 8.43
(1H, d, J = 9.0 Hz, H-8), 7.79 (1H, s, H-1), 7.53 (1H,
d, J = 2.1 Hz, H-5), 7.52 (1H, s, H-1), 7.26 (1H, dd,
J = 9.0, 2.1 Hz, H-7), 4.96 (2H, t, J = 7.4 Hz, H-11),
4.07 (6H, s, OCH3-2 and OCH3), 4.02 (3H, s, OCH3-
6), 3.62 (2H, t, J = 7.4 Hz, H-13), 2.65 (2H, quin,
J = 7.4 Hz, H-12); 13C NMR (100 MHz) d: 162.3,
154.8, 151.9, 151.8, 140.3, 138.3, 133.0, 129.3, 126.2·2,
121.2, 120.1, 117.7, 117.5, 107.4, 107.0, 105.9, 59.7,
57.0, 56.9, 56.3, 32.5, 23.1; EIMS m/z: 360 ([M+ꢀCl],
67), 154 (100). Anal. Calcd for C23H22BrNO3: C,
76.64; H, 6.15; N, 3.89. Found: C, 76.91; H, 6.25; N,
3.96.
4.12. Preparation of dehydrophenanthroindolizidine chlo-
rides 2a and 2b
4.12.1. Dehydroantofine chloride 2a. A solution of anto-
fine 1a (36.3 mg, 1 mmol) in 10 mL CHCl3 was exposed
to light. After one day, a yellow product had formed,
and was filtered and recrystallized from CH3OH to af-
ford pure 2a (28.3 mg, 71.6%): yellow crystals,
4.13.2. Deoxypergularinine bromide 3b. Deoxypergulari-
nine bromide 3b(24.2 mg, 55.1%) was prepared accord-
ing to the above procedure: yellow crystals, >290 ꢁC,
IR (KBr) cmꢀ1: 2965, 1639, 1608, 1516; 1H NMR
(400 MHz, CD3OD) d: 9.51 (1H, s, H-9), 8.31 (1H, s,
H-14), 8.10 (1H, d, J = 9.1 Hz, H-1), 7.13 (1H, s, H-8),
6.95 (1H, dd, J = 9.1, 1.6 Hz, H-2), 6.68 (1H, d,
J = 1.6 Hz, H-4), 6.63 (1H, s, H-5), 4.75 (2H, t,
J = 7.1 Hz, H-11), 3.90 (3H, s, OCH3-7), 3.86 (3H, s,
OCH3-6), 3.81 (3H, s, OCH3-3), 3.43 (2H, t,
J = 7.1 Hz, H-13), 2.55 (2H, quin, J = 7.1 Hz, H-12);
13C NMR (100 MHz) d: 163.3, 151.4, 151.2, 151.0,
140.2, 138.0, 134.8, 128.4, 124.4, 124.1, 120.8, 118.8,
116.8, 116.4, 106.1, 104.9·2, 59.4, 57.4, 56.5, 56.3,
32.2, 23.0; EIMS m/z: 360 ([M+ꢀCl], 55), 154 (100).
Anal. Calcd for C23H22BrNO3: C, 76.64; H, 6.15; N,
3.89. Found: C, 76.32; H, 6.00; N, 3.72.
1
>290 ꢁC, IR (KBr) cmꢀ1: 2968, 1609, 1519; H NMR
(300 MHz, CD3OD) d: 9.89 (1H, s, H-9), 8.74 (1H, s,
H-14), 8.42 (1H, d, J = 8.9 Hz, H-8), 7.77 (1H, s, H-1),
7.52 (1H, d, J = 2.2 Hz, H-5), 7.50 (1H, s, H-1), 7.24
(1H, dd, J = 8.9, 2.2 Hz, H-7), 4.95 (2H, t, J = 7.4 Hz,
H-11), 4.05 (6H, s, OCH3-2 and OCH3), 4.01 (3H, s,
OCH3-6), 3.60 (2H, t, J = 7.4 Hz, H-13), 2.63 (2H, quin,
J = 7.4 Hz, H-12); 13C NMR (100 MHz) d: 161.8, 154.3,
151.6, 151.3, 139.9, 137.9, 132.6, 128.9, 125.7·2, 120.9,
119.6, 117.3, 117.0, 107.0, 106.6, 105.4, 59.5, 56.6,
56.4, 56.0, 32.1, 22.8; EIMS m/z: 360 ([M+ꢀCl], 46),
154 (100). Anal. Calcd for C23H22ClNO3: C, 69.78; H,
5.60; N, 3.54. Found: C, 69.92; H, 5.45; N, 3.36.
4.13.3. In vitro cytotoxicity assay. The sulforhodamine B
assay was used according to the procedures developed
and validated at NCI.18 Doxorubicin was used as the
positive control antitumor drug. The in vitro anticancer
activities are expressed as EC50 values, which are the test
compound concentration (lg/mL) that reduced the cell
number by 50% after 72 h of continuous treatment.
The values were interpolated from dose–response data.
Each test was performed in triplicate with variation less
4.12.2. Deoxypergularinine chloride 2b. Deoxypergulari-
nine chloride 2b (27.8 mg, 70.3%) was prepared accord-
ing to the above procedure: yellow crystals, >290 ꢁC, IR
(KBr) cmꢀ1: 2964, 1608, 1517; 1H NMR (300 MHz,
CD3OD) d: 9.51 (1H, s, H-9), 8.30 (1H, s, H-14), 8.09
(1H, d, J = 9.0 Hz, H-1), 7.13 (1H, s, H-8), 6.94 (1H,
dd, J = 9.0, 1.5 Hz, H-2), 6.68 (1H, d, J = 1.5 Hz, H-
4), 6.61 (1H, s, H-5), 4.74 (2H, t, J = 7.2 Hz, H-11),

C.-R. Su et al. / Bioorg. Med. Chem. 16 (2008) 6233–6241
6241
4. Fu, Y.; Lee, S. K.; Min, H. Y.; Lee, T.; Lee, J.; Cheng, M.;
Kima, S. Bioorg. Med. Chem. Lett. 2007, 17, 97–100.
5. Lee, S. K.; Nam, K. A.; Heo, Y. H. Planta Med. 2003, 69,
21–25.
6. Xi, Z.; Zhang, R.; Yu, Z.; Ouyang, D.; Huang, R. Bioorg.
Med. Chem. Lett. 2005, 15, 2673–2677.
than 5%. The EC50 values determined in each of inde-
pendent tests varied less than 10%. Compound stock
solutions were prepared in DMSO with the final solvent
concentration 61% DMSO (v/v), a concentration with-
out effect on cell replication. The cells were cultured at
37 ꢁC in RPMI-1640 supplemented with 25 mM N-2-
7. Xi, Z.; Zhang, R.; Yu, Z.; Ouyang, D. Bioorg. Med. Chem.
Lett. 2006, 16, 4300–4304.
hydroxyethylpiperazine-N0-2-ethanesulfonic
acid
(HEPES), 2% (w/v) sodium bicarbonate, 10% (v/v) fetal
bovine serum, and 100 lg/mL kanamycin in a humidi-
fied atmosphere containing 5% CO2.
8. Leburn, S.; Couture, A.; Deniau, E.; Grandclaudon, P.
Tetrahedron 1999, 55, 2659–2670.
9. Li, Z.; Jin, Z.; Huang, R. Synthesis 2001, 2365–2378.
10. Kim, X.; Lee, J.; Lee, T.; Park, H. G.; Kim, D. Org. Lett.
2003, 5, 2703–2706.
11. Camacho-Davila, A.; Herndon, J. W. J. Org. Chem. 2006,
71, 6682–6685.
Acknowledgments
12. Furstner, A.; Kennedy, J. W. J. Chem. Eur. J. 2006, 12,
7398–7410.
13. Kim, S.; Lee, T.; Lee, E.; Lee, J.; Fan, G. J.; Lee, S. K.;
Kim, D. J. Org. Chem. 2004, 69, 3144–3149.
14. Ciufolini, M. A.; Roschangar, F. J. Am. Chem. Soc. 1996,
118, 12082–12089.
The authors are grateful for financial support from the
National Science Council, Taiwan, Republic of China
(NSC 95-2323-B-006-003) awarded to T.S. Wu. Thanks
are also due to the support by NIH Grant No. CA-
17625 from the National Cancer Institute awarded to
K.H. Lee. We also thank Dr. T.H. Chuang (National
Cheng Kung University) for his kind discussion.
15. Chuang, T. H.; Lee, S. J.; Yang, C. W.; Wu, P. L. Org.
Biomol. Chem. 2006, 4, 860–867.
16. Bremmer, M. L.; Khatri, N. A.; Weinreb, S. M. J. Org.
Chem. 1983, 48, 3661–3666.
17. Govindachari, T. R.; Viswanathan, N.; Radhakrishan, J.
Indian J. Chem. 1973, 11, 1215–1216.
References and notes
18. Rao, K. V.; Kapicak, L. S. J. Heterocycl. Chem. 1976, 13,
1073–1077.
1. Gellert, E. In Alkaloids: Chemical and Biological Perspec-
tives; Pelletier, S. W., Ed.; Academic Press: New York,
1987; pp 55–132.
2. Damu, A. G.; Kuo, P. C.; Shi, L. S.; Li, C. Y.; Kuoh, C.
S.; Wu, P. L.; Wu, T. S. J. Nat. Prod. 2005, 68, 1071–1075.
3. Staerk, D.; Lykkeberg, A. K.; Christensen, J.; Budnik, B.
A.; Abe, F.; Jaroszewski, J. W. J. Nat. Prod. 2002, 65,
1299–1302.
19. Rubinstein, L. V.; Shoemaker, R. H.; Paull, K. D.; Simon,
R. M.; Tosini, S.; Skehan, P.; Scudiero, D. A.; Monks, M.
R. J. Natl. Cancer Inst. 1990, 82, 1113–1118.
20. Gao, W.; Bussom, S.; Grill, S. P.; Gullen, E. A.; Hu, Y. C.;
Huang, X.; Zhong, S.; Kaczmarek, C.; Gutierrez, J.;
Francis, S.; Baker, D. C.; Yu, S.; Cheng, Y. C. Bioorg.
Med. Chem. Lett. 2007, 17, 4338–4342.