Expedient synthesis and structure-activity relationships of phenanthroindolizidine and phenanthroquinolizidine alkaloids

Article abstract of DOI:10.1039/b516152e

The total synthesis of alkaloids phenanthroindolizidine 1a, tylophorine 1b, and phenanthroquinolizidine 1c, has been achieved in 46%, 49%, and 42% overall yield, respectively, starting from the corresponding phenanthrene-9- carboxaldehyde. Compound 1c exhibited potent inhibition activity in three human cancer cell lines, with IC₅₀ values ranging from 104 to 130 nM. The structure-activity relations of these alkaloids and some of their synthetic intermediates against the three cell lines were also described. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b516152e

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Expedient synthesis and structure–activity relationships of
phenanthroindolizidine and phenanthroquinolizidine alkaloids
Ta-Hsien Chuang,a Shiow-Ju Lee,b Cheng-Wei Yangb and Pei-Lin Wu*a,c
Received 14th November 2005, Accepted 16th December 2005
First published as an Advance Article on the web 18th January 2006
DOI: 10.1039/b516152e
The total synthesis of alkaloids phenanthroindolizidine 1a, tylophorine 1b, and phenanthroquino-
lizidine 1c, has been achieved in 46%, 49%, and 42% overall yield, respectively, starting from the
corresponding phenanthrene-9-carboxaldehyde. Compound 1c exhibited potent inhibition activity in
three human cancer cell lines, with IC50 values ranging from 104 to 130 nM. The structure–activity
relations of these alkaloids and some of their synthetic intermediates against the three cell lines were
also described.
Introduction
Since the first isolation of tylophorine 1b in 1935,1 the phenan-
throindolizidine alkaloids have been a focus of study in the
fields of medicinal and synthetic chemistry. These alkaloids are
well known for their cytotoxic activity, due to the inhibition of
protein2 and nucleic acid3 synthesis.4 In addition, some of these
alkaloids have been shown to possess antiamoebic,5 antibacte-
rial and antifungal activities.6 Recently, we also reported that
phenanthroindolizidines, isolated from the leaves of Ficus septica,
exhibited strong cytotoxic activity against gastric carcinoma
(NUGC-3) and nasopharyngeal carcinoma (HONE-1) cell lines.7
Scheme 1. This method represented a new route for the preparation
The wide range of pharmacological properties that the phenan-
throindolizidine alkaloids exhibit have drawn the attention of
chemists. In the last three decades, for example, there were three
types of methodologies that could give access to tylophorine.
One was by a biogenetically patterned sequence via b-amino
ketone intermediates 2,8 another was by a route involving an
acid-catalyzed cyclization of amino-acids 3,8b,9 and the other was
by the route to tylophorine where both rings of the indolizidine
nucleus were assembled through a single cycloaddition reaction.10
In this paper, we report a convenient synthetic approach to the
pentacyclic alkaloids 1 in high yields. Subsequently, these alkaloids
and synthetic intermediates undergo preliminary screening tests
for their anticancer activities against human cancer cell lines
including breast carcinoma (MCF-7), lung carcinoma (NCI-
H460) and central nervous system carcinoma (SF-268). The
structure–activity relations (SAR) are also discussed.
of the pentacyclic alkaloid in that the nitrogen-containing bicyclic
heterocycles were constructed by double cyclizations. First, the
isocyanate 7, formed by Curtius rearrangement of acyl azide 8,
underwent an electrocyclic reaction followed by hydrogen shift to
yield isoquinolinone 6. Second, the N-haloalkylisoquinolinone 5
proceeded through radical cyclization to afford the pyrrolidine or
piperidine rings 4. Then, hydride reduction would give the target
products 1.
Unsubstituted phenanthroindolizidine 1a was chosen as our
initial target since its simplicity would allow us to test the
feasibility of the approach (Scheme 2). The Wittig reaction
between commercially available phenanthrene-9-carboxaldehyde
10a and (carboethoxymethylene)triphenylphosphorane followed
by hydrolysis directly yielded the desired acid 9a, which upon
purification was found to be exclusively the trans isomer. The
acid 9a was almost quantitatively transformed to the acyl azide
8a on treatment with oxalyl chloride and sodium azide. Since the
azide was unstable under heat, it was immediately used for the
next reaction after purification using a short column. When a
solution of 8a in o-dichlorobenzene containing a catalytic amount
of Hg(OAc)2 was refluxed for 1 h, the Curtius rearrangement,
electrocyclic reaction, and hydrogen rearrangement occurred
to supply the isoquinolinone 6a in 87% yield. Treatment of the
compound 6a with NaH in DMF provided an amide salt, and
subsequent N-alkylation with 1-bromo-3-chloropropane at room
temperature produced a mixture of chloride 5a1 and bromide
5a2 in a 20 : 1 ratio. Even at 0 ◦C, the ratio of the mixed N-
haloalkylisoquinolinones 5a1 and 5a2 was lifted to 50 : 1 in a ca.
95% combined yield. The mixed halides were directly subjected to
Results and discussion
Synthetic results
The synthetic strategy for constructing the phenanthroin-
dolizidines 1a and 1b and phenanthroquinolizidine 1c is shown in
aDepartment of Chemistry, National Cheng Kung University, Tainan, 701,
Taiwan, ROC. E-mail: wupl@mail.ncku.edu.tw; Fax: 886-6-2740552
bDivision of Biotechnology and Pharmaceutical Research, National Health
Research Institutes, Miaoli, 350, Taiwan, ROC
cDepartment of Cosmetic Science, Chung Hwa College of Medical Technol-
ogy, Tainan, 717, Taiwan, ROC
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Scheme 1
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) cat. Hg(OAc)2, o-dichlorobenzene, reflux, 1 h; (d) (i) NaH, DMF; (ii) Br(CH2)nCl, n = 3 or 4, DMF, rt, overnight; (e) NaI,
CH3CN, 125 ◦C, 12 h; (f) AIBN, Bu3SnH, toluene, reflux, 6 h; (g) NaAl(OCH2CH2OMe)2H2, dioxane, reflux, 2 h; (h) NBS, CHCl3, rt, 1 h.
=
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Fig. 1 The key NOESY correlations of 1a (a), 1b (b), and 1c (c).
radical cyclization according to the method of Osornio et al.11 to
give the cyclization adduct 4a in low yield, and there remained a
considerable amount of the starting material 5a1. Because of the
low activity of chloride, the more active iodide was considered
as the radical precursor in the radical cyclization. The mixed
halides were successfully converted to the corresponding iodide
5a3 by halogen exchange with an excess of sodium iodide in
CH3CN. Without further purification, treatment of compound
5a3 and Bu3SnH in boiling toluene, followed by addition of
small incremental amounts of 2,2ꢀ-azobisisobutyronitrile (AIBN,
0.4 mol equiv.) for 6 h, gave phenanthroindolizidione 4a (76%) and
its dehydro derivative 11a (6%). Attempts to avoid the formation of
the undesired product11a by reducing the amount of AIBNcaused
incomplete consumption of the starting material. Reduction of the
lactam 4a with sodium bis(2-methoxyethoxy)aluminium hydride
in refluxing dioxane afforded racemic phenanthroindolizidine 1a
in a 46% overall yield starting from the aldehyde 10a.
On the basis of the above results, we anticipated that tylophorine
1b and phenanthroquinolizidine 1c would be readily available
from 2,3,6,7-tetramethoxyphenanthrene-9-carboxaldehyde 10b.
The same protocol was followed for producing intermediates 9b
(76%), 8b (92%), 6b (85%), 5b1 (96%) and 5c1 (89%) with generally
excellent yields. We were concerned about the intramolecular
radical cyclization to afford the pyrrolidine and piperidine rings,
respectively. Surprisingly, when chloride 5b1 was directly subjected
to the n-Bu3SnH–AIBN reaction condition, the starting material
was completely consumed and generated phenanthroindolizidione
4b (87%) and traces of the dehydro derivative 11b (5%). Comparing
the results of the 5-exo cyclizations of 5a1 and 5b1, the OCH3
groups on the phenanthrene ring might increase the reactivity of
radical cyclization. In contrast to 5b1, the chloride 5c1 showed
inefficient intramolecular cyclization under the same conditions
and led to incomplete consumption of the starting material. It was
thought that this was mainly due to the higher activation energy
for 6-exo ring closure.12 Therefore, phenanthroquinolizidione 4c
(80%) and the oxidative side-product 11c (12%) were obtained via
halogen exchange followed by radical cyclization. Tylophorine 1b
and phenanthroquinolizidine 1c could be afforded by reduction of
the lactams 4b and 4c both in high 98% yields. The overall yields
from the aldehyde 10b to 1b and 1c were calculated to be 49%, and
42%, respectively.
dehydroiminium salts 12a–c which might be obtained by treatment
of 1a–c with N-bromosuccinimide (NBS) in CHCl3.14
The complete assignment of the 1H and 13C NMR signals
of 1a–c was obtained from 2D-NMR spectra, such as COSY,
HMQC, HMBC and NOESY. In 1a, the existence of NOEs
of H-12a (d 2.49) with H-11ax (d 1.94) and H-12eq (d 2.24)
and the absence of NOEs of H-12a with H-9 (d 3.74 and 4.74)
and H-10 (d 2.46 and 3.48) suggested that 1a possessed the cis-
fused indolizidine skeleton with a boat-like conformation in the
six-membered ring and an envelope conformation in the five-
membered ring (Fig. 1a).15 Similarly, the absence of NOEs between
H-12a (d 2.51) and H-9 (d 3.67 and 4.62) and H-10 (d 2.45 and 3.47)
indicated the cis-boat indolizidine ring in 1b (Fig. 1b). In contrast
to the cis-fused phenanthroindolizidine skeleton, the presence of
NOEs between H-13a (d 2.39) and H-9ax (d 3.60), H-10ax (d 2.32)
and H-12ax (d1.49) in 1c suggested a trans-fused quinolizidine
ring adopting a chair-like conformation (Fig. 1c). In addition,
the triplet–quintet–triplet splitting pattern of three mutually
coupled methylenes in 11a–b and 12a–b and the triplet–quintet–
quintet–triplet pattern of four methylenes in 11c and 12c let us
conclude that the pentacyclic rings in dehydrolactams 11a–c and
dehydroiminium salts 12a–c established a planar conformation, in
spite of the five- or six-membered saturated ring.
Biological results
We now describe the biological evaluation of the pentacyclic
alkaloids 1 and some of their analogues 4, 5, 6, 11, and 12, available
by total synthesis. The cytotoxic activities of the synthesized com-
pounds were tested against three human cancer cell lines, MCF-7,
NCI-H460, and SF-268. The results are shown in Table 1. We
started our SAR with tylophorine 1b which showed pronounced
cytotoxicity, with IC50 values of 489 to 1764 nM. A 6 to 20-
fold decrease in cytotoxicity was observed for 1a relative to 1b,
indicating that the methoxyl group on the phenanthrene ring was
of importance. It should be noted that phenanthroquinolizidine
1c exhibited the best activity in the three cell lines ranging from
104 to 130 nM. This revealed that the trans-fused quinolizidine
ring seems to be more active than the cis-fused indolizidine ring.
The lack of either an indolizidine or a quinolizidine ring led
isoquinolinones 5a–c and 6a–b to show no significant activity
in all cell lines. Moreover, comparison of the cytotoxic activities
of lactams 4a–c and the corresponding reduced compounds 1a–c
revealed that the presence of a carbonyl in the bicyclic lactam may
dramatically diminish cytotoxicity. In addition, the almost planar
dehydrolactams 11a–c did not show any activity in our test. This
It should be mentioned here that the phenanthroindolizidine
and phenanthroquinolizidine alkaloids were known to be unstable
when exposed to light.13 We also observed that, after 1 day,
compounds 1a–c in CHCl3 decomposed to a yellow crystalline
solid. The NMR spectral properties showed agreement with
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Table 1 Cytotoxicity of compounds toward several cancer cell linesa
3-(Phenanthren-9-yl)acrylic acid 9a. A mixture of phenan-
threne-9-carboxaldehyde (1.03 g, 5 mmol) and (carboethoxy-
methylene)triphenylphosphorane (2.09 g, 6 mmol) in toluene
(30 cm3) was refluxed under N2 for 4 h. After cooling, the resulting
solution was directly purified by flash chromatography on silica
gel with hexane–EtOAc (1 : 1) as eluent. Recrystallization from
hexane–EtOAc afforded the ethyl ester of 9a (1.21 g, 88%) as
white needles: mp 119–120 ◦C (lit.,16 mp 120–121 ◦C).
A solution of 1 N KOH (10 cm3) was added to a solution of
the ester (1.10 g, 4 mmol) in EtOH (20 cm3) and the reaction
mixture was heated to reflux for 3 h. After cooling, the solution
was evaporated, and the residue was dissolved in water (25 cm3),
acidified with 10% HCl and extracted with EtOAc (5 × 20 cm3).
The combined extracts were dried with anhydrous MgSO4, filtered,
and evaporated under reduced◦pressure to give 9a (0.96 g, 97%)
as white crystals: mp 224–225 C (from EtOAc) (lit.,17 mp 230–
233 ◦C); (found: C, 82.27; H, 4.88. Calcd for C17H12O2: C, 82.24; H,
4.87%); mmax(KBr)/cm−1 3000 (br), 1694 and 1630; dH(300 MHz;
DMSO-d6; Me4Si) 6.69 (1H, d, J 15.7), 7.72 (4H, m), 8.07 (1H, d,
J 7.5), 8.23 (1H, m), 8.30 (1H, s), 8.40 (1H, d, J 15.7), 8.84 (1H, d,
J 8.1), 8.91 (1H, m) and 12.57 (1H, br s); dC(75 MHz; DMSO-d6;
Me4Si) 122.6, 122.9, 123.6, 124.0, 126.5, 127.2, 127.3, 127.5, 128.0,
129.3, 129.4, 129.9, 130.0, 130.4, 130.8, 140.8 and 167.4; m/z (EI)
248 (M+, 31%), 203 (90), 202 (100) and 176 (17).
IC50/nMb
Compound
MCF-7
NCI-H460
SF-268
1a
9997 408
489 45
10 231 632
584 39
10 811 1213
1764 105
1b
1c
104
>50 000
>50 000
7
109
>50 000
>50 000
9
130
>50 000
>50 000
3
4a
4b
4c
41 603 2815
13 687 1233
11 435 222
>50 000
>50 000
>50 000
>50 000
>50 000
>50 000
>50 000
24 144 471
17 229 975
17 462 1818
>50 000
>50 000
>50 000
>50 000
>50 000
>50 000
>50 000
34 070 607
17 849 948
7567 627
>50 000
>50 000
>50 000
>50 000
>50 000
>50 000
>50 000
5a1
5a2
5b1
5c1
6a
6b
11a
11b
11c
12a
12b
12c
8275 74
>50 000
13 483 2615
>50 000
23 038 2435
4440 164
>50 000
11 878 2212
2764 296
a MCF-7 = human breast carcinoma; NCI-H460 = human lung car-
cinoma; SF-268 = human central nervous system carcinoma. b Values
are means SD, where SD = standard deviation; all experiments were
independently performed at least three times.
probably resulted from the rigid indolizidinone or quinolizidinone
structure. Therefore, the planar dehydroiminium salts 12a–c with
no carbonyl group showed substantial loss of activity.
3-(2,3,6,7-Tetramethoxyphenanthren-9-yl)acrylic acid 9b. The
analogous procedure for the preparation of acid 9a was used.
The aldehyde 10b18 (1.63 g, 5 mmol) gave 9b (1.40 g, 76%) as
pale yellow crystals: mp 282–284 ◦C (from o-dichlorobenzene);
(found: C, 68.03; H, 5.43. Calcd for C21H20O6: C, 68.47; H, 5.47%);
mmax(KBr)/cm−1 3000 (br), 1686 and 1616; dH(300 MHz; DMSO-
d6; Me4Si) 3.91 (3H, s), 3.97 (3H, s), 4.05 (6H, s), 6.59 (1H, d,
J 15.6), 7.47 (2H, s), 8.00 (1H, s), 8.06 (1H, s), 8.08 (1H, s), 8.36
(1H, d, J 15.6) and 12.46 (1H, br s); dC(75 MHz; DMSO-d6; Me4Si)
55.4, 55.9, 103.5, 104.0, 104.3, 108.9, 120.8, 123.7, 123.9, 124.4,
125.2, 125.3, 127.0, 141.5, 148.7, 148.8, 149.1, 150.1 and 167.6;
m/z (EI) 368 (M+, 100%), 279 (13), 249 (13), 207 (14), 176 (20)
and 163 (24).
Conclusions
In conclusion, the indolizidine and quinolizidine rings were
constructed by thermal and radical cyclizations. By the protocol
outlined above, tylophorine 1b and the modified analogues 1a
and 1c were synthesized in high yields. The SAR of phenan-
throindolizidine and phenanthroquinolizidine skeletons toward
inhibition of three cancer lines, MCF-7, NCI-H460, and SF-
268, revealed that the more rigid bicyclic heterocycles 11a–
c and 12a–c showed a remarkable decrease in cytotoxicity. A
potency improvement was observed by the introduction of a
methoxyl functionality on the phenanthrene ring. A trans-fused
quinolizidine ring could further enhance the cytotoxic activities.
Compound 1c, for instance, is clearly a promising anticancer agent
for further study.
3-(Phenanthren-9-yl)acryloyl azide 8a. A mixture of acid 9a
(1.24 g, 5 mmol) and oxalyl chlori◦de (1.27 g, 10 mmol) in toluene
(50 cm3) was heated for 5 h at 80 C. After cooling, the resulting
mixture was concentrated under reducedpressure toafford the acyl
chloride quantitatively. The acyl chloride was added immediately
into a suspension of NaN3 (0.98 g, 15 mmol) in dry acetone
(30 cm3) on an ice bath. The reaction mixture was stirred gently
for 2 h at room temperature and filtered.19 The solvent was
evaporated in vacuo, and the residue was purified by short column
chromatography over silica gel eluting with CHCl3–hexane (2 : 1)
to yield the pure acyl azide 8a (1.27 g, 93%) as a pale yellow solid:
mmax(KBr)/cm−1 2145 and 1682; dH(300 MHz; CDCl3; Me4Si) 6.62
(1H, d, J 15.5), 7.68 (4H, m), 7.93 (1H, d, J 7.7), 8.03 (1H, s), 8.20
(1H, d, J 7.2), 8.61 (1H, d, J 15.5), 8.68 (1H, d, J 8.2) and 8.76 (1H,
d, J 7.5); dC(75 MHz; CDCl3; Me4Si) 121.9, 122.6, 123.3, 124.0,
127.0, 127.1, 127.2, 127.3, 128.1, 129.4, 129.8, 130.1, 130.4, 130.9,
131.4, 144.2 and 171.9; m/z (EI) 245.0841 (M+ − N2. C17H11NO
requires 245.0841), 202 (100%), 189 (33) and 176 (24).
Experimental
General
Melting points were taken on a Buchi 535 melting-point apparatus
and were not corrected. Infrared spectra were measured on a
Nicolet Magna FT-IR spectrometer as either thin film or solid dis-
persion in KBr. Nuclear magnetic resonance spectra were recorded
on Bruker Avance-300 and AMX-400 FT-NMR spectrometers; all
chemical shifts were reported in ppm from tetramethylsilane as an
internal standard. Mass spectra were obtained on a VG 70-250S
spectrometer. Elemental analyses were performed on a Heraeus
CHN-RAPID elemental analyzer. Column chromatography was
carried out using 70–230 mesh silica gel.
3-(2,3,6,7-Tetramethoxyphenanthren-9-yl)acryloyl azide 8b.
The analogous procedure for the preparation of azide 8a was
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used. The acid 9b (1.84 g, 5 mmol) gave 8b (1.80 g, 92%) as a
pale yellow solid: mmax(KBr)/cm−1 2140 and 1677; dH(300 MHz;
CDCl3; Me4Si) 4.04 (3H, s), 4.07 (3H, s), 4.13 (6H, s), 6.56 (1H, d,
J 15.4), 7.20 (1H, s), 7.41 (1H, s), 7.72 (1H, s), 7.78 (1H, s), 7.84
(1H, s) and 8.50 (1H, d, J 15.4); dC(75 MHz; CDCl3; Me4Si) 56.0,
102.6, 103.2, 104.0, 108.7, 120.4, 124.4, 124.7, 125.5, 126.1, 127.2,
129.3, 129.6, 129.9, 132.4, 134.1, 138.0 and 162.3; m/z (EI) 323
([M + 2]+, 30%), 321.0921 (M+. C20H16ClNO requires 321.0920),
286 (100), 245 (60), 228 (77) and 202 (41); for 5a2: white solid: mp
92–93 ◦C; mmax(KBr)/cm−1 1645; dH(300 MHz; CDCl3; Me4Si) 2.49
(2H, quintet, J 6.3), 3.49 (2H, t, J 6.3), 4.32 (2H, t, J 6.3), 7.36
(1H, d, J 7.5), 7.55 (1H, d, J 7.5), 7.72 (4H, m), 8.44 (1H, d, J 8.0),
8.72 (2H, t, J 8.0) and 8.72 (1H, dd, J 8.0 and 2.0); dC(75 MHz;
CDCl3; Me4Si) 30.7, 31.2, 49.0, 101.3, 119.1, 122.2, 123.2, 124.5,
126.9, 127.0, 127.2, 127.7, 127.9, 129.2, 129.6, 129.9, 132.3, 134.1,
138.0 and 162.2; m/z (EI) 367 ([M + 2]+, 39%), 365.0418 (M+.
C20H16BrNO requires 365.0415), 286 (100), 259 (39), 245 (49), 228
(49), 202 (45) and 189 (50).
144.5, 149.2, 149.4, 150.6 and 172.0; m/z (EI) 365.1267 (M+
−
N2. C21H19NO5 requires 365.1263), 306 (15%) and 164 (10).
2H-2-Aza-triphenylen-1-one 6a. A mixture of azide 8a (1.1 g,
4 mmol) and Hg(OAc)2 (31.9 mg, 0.1 mmol) in o-dichlorobenzene
(50 cm3) was refluxed for 1 h. After cooling, the compound 6a was
isolated by precipitation and trituration with hexane to give 6a
◦
(0.86 g, 87%) as pale yellow needles: mp >300 C (from EtOH);
6,7,10,11-Tetramethoxy-2-(3-chloropropyl)-2-aza-triphenylen-1-
one 5b1. The analogous procedure for the preparation of 5a1
was used. The compound 6b (365 mg, 1mmol) gave pure 5b1
(423 mg, 96%) as pale yellow crystals: mp 211–212 ◦C (from
CHCl3–hexane); (found: C, 65.52; H, 5.75; N, 3.02. Calcd for
C24H24ClNO5: C, 65.23; H, 5.47; N, 3.17%); mmax(KBr)/cm−1 1645;
dH(300 MHz; CDCl3; Me4Si) 2.41 (2H, quintet, J 6.1), 3.64 (2H,
t, J 6.1), 4.09 (3H, s), 4.13 (3H, s), 4.14 (3H, s), 4.16 (3H, s), 4.32
(2H, t, J 6.1), 7.20 (1H, d, J 7.5), 7.46 (1H, d, J 7.5), 7.70 (1H, s),
7.79 (1H, s), 7.80 (1H, s) and 10.05 (1H, s); dC(75 MHz; CDCl3;
Me4Si) 31.1, 42.0, 47.6, 55.7, 55.8, 55.9, 101.3, 102.6, 103.0, 104.8,
108.9, 117.3, 120.8, 124.0, 124.5, 126.9, 132.6, 135.6, 148.5, 148.6,
148.7, 150.8 and 162.4; m/z (EI) 443 ([M + 2]+, 37%), 441 (M+,
100) and 405 (23).
(found: C, 83.05; H, 4.53; N, 5.71. Calcd for C17H11NO: C, 83.25;
H, 4.52; N, 5.71%); mmax(KBr)/cm−1 3135 and 1630; dH(300 MHz;
DMSO-d6; Me4Si) 7.56 (2H, m), 7.70 (2H, m), 7.76 (1H, t, J 7.8),
7.85 (1H, t, J 7.8), 8.70 (1H, d, J 7.8), 8.88 (2H, m), 10.32 (1H, m)
and 11.93 (1H, br s); dC(75 MHz; DMSO-d6; Me4Si) 100.6, 118.1,
122.7, 123.5, 125.4, 126.8, 127.2, 127.4, 127.6, 128.8, 129.6, 131.6,
131.9, 138.8 and 162.6; m/z (EI) 245 (M+, 64%), 216 (26) and 189
(100).
6,7,10,11-Tetramethoxy-2H-2-aza-triphenylen-1-one 6b. The
analogous procedure for the preparation of 6a was used. The
azide 8b (1.97 g, 5 mmol) gave 6b (1.56 g, 85%) as tan needles: mp
>300 ◦C; (found: C, 68.75; H, 5.30; N, 3.75. Calcd for C21H19NO5:
C, 69.03; H, 5.24; N, 3.83%.); mmax(KBr)/cm−1 3115 and 1631;
dH(300 MHz; DMSO-d6; Me4Si) 3.92 (3H, s), 4.01 (3H, s), 4.05
(3H, s), 4.09 (3H, s), 7.48 (2H, m), 7.94 (1H, s), 8.05 (2H, s), 10.05
(1H, s) and 11.63 (1H, d, J 4.4); dC(75 MHz; DMSO-d6; Me4Si)
55.2, 55.7, 55.9, 101.1, 104.1, 104.2, 106.0, 108.7, 116.6, 120.9,
123.6, 124.0, 126.5, 129.9, 136.5, 148.3, 148.5, 148.8, 151.1 and
162.8; m/z (EI) 365 (M+, 100%) and 332 (18).
6,7,10,11-Tetramethoxy-2-(4-chlorobutyl)-2-aza-triphenylen-1-
one 5c1. The analogous procedure for the preparation of 5a1 was
used. The compound 6b (365 mg, 1mmol) reacted with 1-bromo-
4-chlorobutane (686 mg, 4 mmol) and gave pure 5c1 (405 g, 89%)
as pale yellow crystals: mp 184–185 ◦C (from EtOAc); (found: C,
65.40; H, 5.82; N, 3.02. Calcd for C25H26ClNO5: C, 65.86; H, 5.75;
N, 3.07%); mmax(KBr)/cm−1 1645; dH(300 MHz; CDCl3; Me4Si)
1.91 (2H, quintet, J 6.1), 2.05 (2H, quintet, J 6.1), 3.61 (2H, t, J
6.1), 4.14 (14H, m), 7.20 (1H, d, J 7.3), 7.37 (1H, d, J 7.3), 7.70
(1H, s), 7.80 (2H, s) and 10.08 (1H, s); dC(75 MHz; CDCl3; Me4Si)
26.7, 29.7, 44.4, 49.3, 55.7, 55.8, 55.9, 101.5, 102.7, 103.1, 104.9,
109.1, 117.5, 120.9, 124.1, 124.7, 127.0, 132.0, 135.5, 148.5, 148.7,
148.8, 150.9 and 162.4; m/z (EI) 457 ([M + 2]+, 38%), 455 (M+,
100) and 421 (49).
2-(3-Chloropropyl)-2-aza-triphenylen-1-one 5a1 and 2-(3-
bromopropyl)-2-aza-triphenylen-1-one 5a2. To a suspension of
NaH (60% dispersion in oil, 80 mg, 2 mmol) in DMF (5 cm3),
cooled in an ice bath, a solution of 6a (245 mg, 1 mmol) in DMF
(15 cm3) was added with stirring at a rate such as to maintain
gentle evolution of hydrogen. After the addition was complete,
the reaction mixture was stirred at room temperature for 30 min.
This mixture was added dropwise to a solution of 1-bromo-3-
chloropropane (628 mg, 4 mmol) and DMF (5 cm3) with stirring.
This mixture was stirred at room temperature overnight. The
solvent was evaporated in vacuo, and water (10 cm3) was then
added. The mixture was extracted with CHCl3, and the combined
extracts were washed with water, dried with anhydrous MgSO4,
and filtered. The filtrate was concentrated, and the residue was
purified by column chromatography over silica gel eluting with
pureCHCl3 to give a 20 : 1 mixture of chloride5a1 and bromide5a2
(95% combined yield). The mixture was rechromatographed with
CHCl3–hexane (5 : 1) to give a material enriched in 5a1 and a little
of pure 5a2 for spectral and mass analysis. For 5a1: mmax(KBr)/cm−1
1646; dH(300 MHz; CDCl3; Me4Si) 2.39 (2H, quintet, J 6.3), 3.62
(2H, t, J 6.3), 4.30 (2H, t, J 6.3), 7.32 (1H, d, J 7.4), 7.49 (1H, d,
J 7.4), 7.71 (4H, m), 8.41 (1H, d, J 8.1), 8.69 (2H, t, J 8.1) and
10.31 (1H, d, J 8.1); dC(75 MHz; CDCl3; Me4Si) 31.1, 42.1, 48.0,
101.3, 119.2, 122.3, 123.3, 124.6, 126.9, 127.1, 127.3, 127.8, 127.9,
11,12,12a,13-Tetrahydro-10H-9a-aza-cyclopenta[b]triphenylen-
9-one 4a and 11,12-dihydro-10H-9a-aza-cyclopenta[b]triphenylen-
9-one 11a. A solution of chloride 5a1 (161 mg, 0.5 mmol) and
NaI (375 mg, 2.5 mmol) in dry CH3CN (10 cm3) was placed in
◦
a sealed tube and then heated at 125 C for 12 h. After cooling,
the reaction mixture was filtered, and concentrated in vacuo to
yield iodide 5a3 quantitatively. Subsequently, a solution of AIBN
(33 mg, 0.2 mmol) in toluene (2.4 cm3) was added dropwise (syringe
pump) to a degassed solution of iodide 5a3 (207 mg, 0.5 mmol)
and n-Bu3SnH (175 mg, 0.6 mmol) in refluxing toluene (25 cm3)
for 6 h. The reaction mixture was then cooled and the solvent
removed under reduced pressure. The residue was triturated with
hexane (3 × 2 cm3) and purified by column chromatography over
silica gel eluting with CHCl3–hexane (5 : 1) to give 4a (109 mg,
76%) as white crystals and 11a (8 mg, 6%) as a white solid. For
4a: mp 163–164 ◦C (from CHCl3–hexane); (found: C, 83.39; H,
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6.01; N, 4.85. Calcd for C20H17NO: C, 83.59; H, 5.96; N, 4.87%);
mmax(KBr)/cm−1 1636; dH(300 MHz; CDCl3; Me4Si) 1.80 (1H, m),
1.85 (1H, m), 1.96 (1H, m), 2.31 (1H, m), 2.82 (1H, dd, J 15.6
and 13.8), 3.57 (1H, dd, J 15.6 and 3.8), 3.77 (2H, m), 3.79 (1H,
m), 7.62 (4H, m), 7.98 (1H, d, J 8.1), 8.63 (1H, d, J 8.1), 8.66
(1H, d, J 8.1) and 9.30 (1H, d, J 8.1); dC(75 MHz; CDCl3; Me4Si)
23.5, 32.2, 33.6, 45.2, 55.0, 122.2, 123.1, 124.6, 125.3, 126.3, 126.8,
127.0, 127.7, 127.9, 128.9, 129.1, 129.9, 131.7, 135.6 and 163.8;
m/z (EI)◦287 (M+, 82%), 218 (100) and 190 (84); for 11a: mp
230–232 C; mmax(KBr)/cm−1 1650; dH(300 MHz; CDCl3; Me4Si)
2.33 (2H, quintet, J 7.6), 3.31 (2H, t, J 7.6), 4.38 (2H, t, J 7.6),
7.34 (1H, s), 7.71 (4H, m), 8.46 (1H, d, J 8.2), 8.69 (1H, d, J
8.2), 8.73 (1H, d, J 8.2) and 10.39 (1H, d, J 8.2); dC(75 MHz;
CDCl3; Me4Si) 21.4, 32.1, 49.2, 96.2, 116.8, 122.1, 123.3, 124.7,
126.4, 126.9, 127.7, 129.0, 130.4, 132.5, 139.0, 146.1 and 162.0;
m/z (EI) 285.1152 (M+. C20H15NO requires 285.1154) and
189 (12%).
mmax(KBr)/cm−1 1636; dH(300 MHz; CDCl3; Me4Si) 1.92 (2H,
quintet, J 6.4), 2.08 (2H, quintet, J 6.4), 3.03 (2H, t, J 6.4), 4.12
(3H, s), 4.13 (3H, s), 4.15 (3H, s), 4.16 (3H, s), 4.23 (2H, t, J 6.4),
7.02 (1H, s), 7.73 (1H, s), 7.84 (2H, s) and 10.09 (1H, s); dC(75 MHz;
CDCl3; Me4Si) 19.2, 22.8, 29.4, 42.1, 56.0 (4 × C), 101.1, 103.1,
103.5, 105.2, 109.0, 115.0, 120.9, 123.6, 125.0, 127.3, 135.3, 141.9,
148.3, 148.7, 148.9, 151.0 and 163.4; m/z (EI) 419.1736 (M+.
C25H25NO5 requires 419.1733), 404 (17%), 376 (18), 322 (14)
and 318 (13).
9,10,11,12,12a,13-Hexahydro-9a-aza-cyclopenta[b]triphenylene
1a. To a solution of the lactam 4a (29 mg, 0.1 mmol) in dry
dioxane (5 cm3) was added a 3.5 M solution of sodium bis(2-
methoxyethoxy)aluminium hydride in toluene (0.4 cm3, 1.4 mmol)
and the mixture was refluxed for 2 h in the dark. After evaporation
of the solvents, the residue was diluted with water (10 cm3)
and then basified with 10% aqueous NaOH. The mixture was
extracted with CHCl3 (5 × 15 cm3), and the combined extracts were
washed with water, dried with anhydrous MgSO4, and filtered. The
filtrate was concentrated, and the residue was purified by column
chromatography over silica gel eluting with CHCl3–MeOH (50 : 1)
to give 1a (25 mg, 92%) as white powder: mp 169–170 ◦C (decomp.)
(lit.,21 mp 170 ◦C); mmax(KBr)/cm−1 1606 and 1495; dH(300 MHz;
CDCl3; Me4Si) 1.78 (1H, m, H-12ax), 1.94 (1H, m, H-11ax), 2.02
(1H, m, H-11eq), 2.24 (1H, m, H-12eq), 2.46 (1H, m, H-10ax),
2.49 (1H, m, H-12a), 2.99 (1H, dd, J 16.0 and 10.5, H-13ax), 3.48
(2H, m, H-10eq and H-13eq), 3.74 (1H, d, J 15.2, H-9ax), 4.74
(1H, d, J 15.2, H-9eq), 7.62 (4H, m, H-2, H-3, H-6, and H-7),
7.92 (1H, dd, J 7.8 and 1.5, H-8), 8.04 (1H, dd, J 6.0 and 3.3,
H-1) and 8.70 (2H, m, H-4 and H-5); dC(75 MHz; CDCl3; Me4Si)
21.6 (C-11), 31.3 (C-12), 33.7 (C-13), 53.9 (C-9), 55.1 (C-10), 60.1
(C-12a), 122.6 (C-5), 122.8 (C-8), 122.9 (C-9), 123.4 (C-1), 125.8
(C-6), 125.9 (C-3), 126.7 (C-2 and C-7), 128.4 (C-8b), 128.8 (C-
13a), 129.3 (C-4b), 129.5 (C-4a), 130.0 (C-8a) and 131.5 (C-13b);
m/z (EI) 273.1517 (M+. C20H19N requires 273.1517), 245 (17%),
228 (27), 202 (100) and 189 (38).
Tylophorin-9-one 4b and 2,3,6,7-tetramethoxy-11,12-dihydro-
10H-9a-aza-cyclopenta[b]triphenylen-9-one 11b. By the analo-
gous procedure for the radical cyclization of iodide 5a3, chloride
5b1 (221 mg, 0.5 mmol) gave 4b (177 mg, 87%) as white crystals
and 11b (10 mg, 5%) as a pale yellow solid. For 4b: mp 284–286 ◦C
(from CHCl3–hexane) (lit.,20 mp 280–281 ◦C); (found: C, 70.69; H,
6.40; N, 3.27. Calcd for C24H25NO5: C, 70.74; H, 6.18; N, 3.44%);
mmax(KBr)/cm−1 1622; dH(300 MHz; CDCl3; Me4Si) 1.93 (1H, m),
1.96 (1H, m), 2.17 (1H, m), 2.43 (1H, m), 2.96 (1H, dd, J 15.4 and
13.4), 3.61 (1H, dd, J 15.4 and 4.0), 3.84 (2H, m), 3.87 (1H, m), 4.06
(3H, s), 4.08 (3H, s), 4.12 (3H, s), 4.15 (3H, s), 7.36 (1H, s), 7.79
(1H, s), 7.84 (1H, s) and 9.03 (1H, s); dC(75 MHz; CDCl3; Me4Si)
23.4, 32.4, 33.8, 45.2, 55.1, 55.8, 102.2, 102.9, 104.7, 107.9, 122.3,
123.0, 124.2, 124.3, 126.5, 133.2, 148.5, 148.6, 148.8, 150.1 and
164.6; m/z (EI) 407 (M+, 100%), 338 (24), 310 (20) and 295 (16);
for 11b: mp >300 ◦C; mmax(KBr)/cm−1 1647; dH(300 MHz; CDCl3;
Me4Si) 2.32 (2H, quintet, J 7.5), 3.31 (2H, t, J 7.5), 4.10 (3H, s),
4.13 (3H, s), 4.15 (3H, s), 4.16 (3H, s), 4.37 (2H, t, J 7.5), 7.15 (1H,
s), 7.71 (1H, s), 7.81 (2H, s) and 10.15 (1H, s); dC(75 MHz; CDCl3;
Me4Si) 21.6, 31.9, 49.1, 55.9, 96.3, 103.0, 103.4, 105.3, 108.9, 115.4,
121.4, 123.5, 125.1, 127.2, 136.9, 144.6, 148.3, 148.7, 148.9, 151.0
and 162.3; m/z (EI) 405.1579 (M+. C24H23NO5 requires 405.1576),
362 (14%) and 304 (13).
Tylophorine 1b. The analogous procedure for the preparation
of 1a was used. The lactam 4b (41 ◦mg, 0.1 mmol) gave 1b (39 mg,
98%) as white powder: mp 270 C (decomp.) (lit.,22 mp 282–
284 ◦C); mmax(KBr)/cm−1 1618 and 1515; dH(300 MHz; CDCl3;
Me4Si) 1.78 (1H, m, H-12ax), 1.94 (1H, m, H-11ax), 2.03 (1H,
m, H-11eq), 2.29 (1H, m, H-12eq), 2.45 (1H, m, H-10eq), 2.51
(1H, m, H-12a), 2.92 (1H, dd, J 15.6 and 10.7, H-13ax), 3.36 (1H,
dd, J 15.6 and 2.3, H-13eq), 3.47 (1H, t, J 8.4, H-10ax), 3.67
(1H, d, J 14.5, H-9eq), 4.05 (6H, s, 2-OCH3 and 7-OCH3), 4.11
(6H, s, 3-OCH3 and 6-OCH3), 4.62 (1H, d, J 14.5, H-9ax), 7.15
(1H, s, H-8), 7.31 (1H, s, H-1) and 7.82 (2H, s, H-4 and H-5);
dC(75 MHz; CDCl3; Me4Si) 21.6 (C-11), 31.2 (C-12), 33.7 (C-13),
53.9 (C-9), 55.1 (C-10), 55.8 (2-OCH3), 55.9 (7-OCH3), 56.0 (3-
OCH3 and 6-OCH3), 60.2 (C-12a), 103.1 (C-8), 103.3 (C-4), 103.4
(C-5), 104.0 (C-1), 123.4 (C-4b), 123.6 (C-4a), 124.3 (C-13a), 125.8
(C-8a), 125.9 (C-8b), 126.2 (C-13b), 148.4 (C-3), 148.5 (C-6) and
148.7 (C-2 and C-7); m/z (EI) 393.1943 (M+. C24H27NO4 requires
393.1940), 324 (100%) and 309 (12).
2,3,6,7-Tetramethoxy-10,11,12,13,13a,14-hexahydro-9a-aza-
benzo[b]triphenylen-9-one 4c and 2,3,6,7-tetramethoxy-10,11,12,13-
tetrahydro-9a-aza-benzo[b]triphenylen-9-one 11c. The analogous
procedure for the preparation of 4a was used. The compound 5c1
(228 mg, 0.5 mmol) gave 4c (168 mg, 80%) as white crystals a◦nd
11c (25 mg, 12%) as a pale yellow solid. For 4c: mp 190–191 C
(from CHCl3–hexane); (found: C, 71.24; H, 6.46; N, 3.32. Calcd
for C25H27NO5: C, 70.92; H, 6.43; N, 3.32%); mmax(KBr)/cm−1 1621;
dH(300 MHz; CDCl3; Me4Si) 1.52 (1H, m), 1.64 (2H, m), 1.92 (2H,
m), 2.04 (1H, m), 2.90 (1H, td, J 12.6 and 1.5), 3.05 (1H, dd, J
16.3 and 10.9), 3.46 (1H, dd, J 16.3 and 5.0), 3.61 (1H, m), 4.06
(3H, s), 4.10 (3H, s), 4.12 (3H, s), 4.14 (3H, s), 4.72 (1H, br d,
J 12.6), 7.34 (1H, s), 7.79 (1H, s), 7.82 (1H, s) and 9.39 (1H,
s); dC(75 MHz; CDCl3; Me4Si) 23.0, 24.7, 33.0, 42.8, 52.8, 56.0,
102.5, 103.2, 104.7, 108.7, 120.2, 122.9, 124.6, 124.8, 126.9, 133.4,
148.5, 148.9, 149.0, 150.6 and 167.6; m/z (EI) 421 (M+, 100%),
338 (36), 310 (19), 276 (19) and 203 (39); for 11c: mp 122–123 ◦C;
2,3,6,7-Tetramethoxy-10,11,12,13,13a,14-hexahydro-9H-9a-aza-
benzo[b]triphenylene 1c. The analogous procedure for the
preparation of 1a was used. The lactam 4c (42 mg, 0.1 mmol)
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gave 1c (40 mg, 98%) as white powder: mp 245–247 ◦C (decomp.);
mmax(KBr)/cm−1 1615 and 1514; dH(300 MHz; CDCl3; Me4Si) 1.49
(1H, m, H-12ax), 1.55 (1H, m, H-13ax), 1.81 (2H, m, H-11), 1.88
(1H, m, H-12eq), 2.05 (1H, br d, J 11.2, H-13eq), 2.32 (1H, m,
H-10ax), 2.39 (1H, m, H-13a), 2.92 (1H, dd, J 16.6 and 10.7,
H-14ax), 3.12 (1H, br d, J 16.6, H-14eq), 3.29 (1H, br d, J 10.7,
H-10eq), 3.60 (1H, br d, J 15.3, H-9ax), 4.04 (3H, s, 7-OCH3),
4.05 (3H, s, 2-OCH3), 4.11 (6H, s, 3-OCH3 and 6-OCH3), 4.36
(1H, br d, J 15.3, H-9eq), 7.13 (1H, s, H-8), 7.26 (1H, s, H-1)
and 7.82 (2H, s, H-4 and H-5); dC(75 MHz; CDCl3; Me4Si) 24.3
(C-12), 25.9 (C-11), 33.7 (C-13), 34.8 (C-14), 56.0 (2-OCH3,
3-OCH3, 6-OCH3 and 7-OCH3), 56.2 (C-9), 56.3 (C-10), 57.6
(C-13a), 103.0 (C-8), 103.5 (C-4 and C-5), 103.9 (C-1), 123.3
(C-14b), 123.5 (C-8b), 123.9 (C-14a), 124.9 (C-4b), 125.2 (C-4a
and C-8a), 148.4 (C-3 and C-6) and 148.7 (C-2 and C-7); m/z
(EI) 407.2100 (M+. C25H29NO4 requires 407.2097), 324 (100%)
and 294 (22).
H460 were maintained in RPMI medium (ICN) supplemented
with 10% fetal bovine serum (Biological Industries Inc.) and were
seeded in 96 well plates and incubated in a CO2 incubator at 37 ◦C
for 24 h. The seeding numbers were 6500, 7500, and 2500 per
well, respectively. The cells were treated with at least ten different
concentrations of test compounds in a CO2 incubator for 72 h.
The number of viable cells was estimated using the tetrazolium dye
reduction assay (MTS assay), and the experiment was performed
as the manufacturer recommended (Promega, Madison, WI). The
absorbance was measured at 490 nm on a Wallac 1420 VICTOR2
Multilabel Counter (Perkin-Elmer, Boston, MA). The results of
these assays were used to obtain the dose–response curves from
which the IC50 values were determined. An IC50 value represents
the concentration (nM) of the test compound at which a 50%
cell growth inhibition after 3 days of incubation is produced.
The values represent averages of three or more independent
experiments, each with duplicate samples.
11,12-Dihydro-10H-9a-azoniacyclopenta[b]triphenylene 12a.
To a solution of phenanthroindolizidine 1a (27 mg, 0.1 mmol)
in CHCl3 (10 cm3) was added NBS (150 mg, 0.4 mmol) in
small portions with stirring. The solution turned orange-red and
began to deposit an orange crystalline solid. After 1 h following
the addition, the solid was filtered and purified by column
chromatography over Al2O3 eluting with CHCl3–MeOH (10 : 1)
to give 12a (27 mg, 78%) as a yellow solid: mp 219 ◦C (decomp.);
mmax(KBr)/cm−1 1640 and 1618; dH(300 MHz; DMSO-d6; Me4Si)
2.55 (2H, quintet, J 7.8), 3.64 (2H, t, J 7.8), 5.02 (2H, t, J 7.8),
7.93 (3H, m), 8.04 (1H, t, J 7.6), 8.97 (4H, m), 9.40 (1H, s) and
10.58 (1H, s); dC(75 MHz; DMSO-d6; Me4Si) 21.9, 31.4, 58.7,
117.4, 123.8, 124.2, 124.3, 124.9, 125.3, 125.8, 126.1, 128.8, 128.9,
129.8, 130.2, 132.4, 132.5, 139.5, 140.3 and 152.9; m/z (FAB)
270.1280 (M+ − Br. C20H16N requires 270.1283).
Acknowledgements
The authors would like to thank the National Science Council of
the Republic of China for the financial support (NSC 93-2113-B-
006-006).
References
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Dehydrotylophorine 12b. The analogous procedure for the
preparation of 12a was used. Phenanthroindolizidine 1b (39 mg,
0.1 mmol) gave 12b (24 mg, 51%) as a yellow solid: mp >300 C
◦
(decomp.); mmax(KBr)/cm−1 1632 and 1611; dH(300 MHz; DMSO-
d6; Me4Si) 2.50 (2H, quintet, J 7.3), 3.61 (2H, t, J 7.3), 4.07 (6H, s),
4.11 (3H, s), 4.14 (3H, s), 4.99 (2H, t, J 7.3), 8.09 (2H, s), 8.26 (1H,
s), 8.29 (1H, s), 9.31 (1H, s) and 10.50 (1H, s); dC(75 MHz; DMSO-
d6; Me4Si) 22.2, 31.3, 56.4, 58.3, 105.0, 105.2, 106.7, 116.6, 119.0,
119.8, 123.7, 124.5, 128.1, 138.6, 138.7, 149.6, 149.9, 150.4, 151.2
and 153.3; m/z (FAB) 390.1704 (M+ − Br. C24H24NO4 requires
390.1705).
5 K. K. Bhutani, G. L. Sharma and M. Ali, Planta Med., 1987, 53,
532–536.
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1075.
2,3,6,7-Tetramethoxy-10,11,12,13-tetrahydro-9a-azoniabenzo-
[b]triphenylene 12c. The analogous procedure for the preparation
of 12a was used. Phenanthroquinolizidine 1c (41 mg, 0.1 mmol)
gave 12c (30 mg, 62%) as a yellow solid: mp 268 ◦C (decomp.);
mmax(KBr)/cm−1 1636 and 1610; dH(300 MHz; DMSO-d6; Me4Si)
2.03 (2H, quintet, J 6.3), 2.19 (2H, quintet, J 6.3), 3.38 (2H, t, J
6.3), 4.10 (6H, s), 4.11 (3H, s), 4.14 (3H, s), 4.81 (2H, t, J 6.3), 8.04
(2H, s), 8.24 (1H, s), 8.26 (1H, s), 9.15 (1H, s) and 10.24 (1H, s);
dC(75 MHz; DMSO-d6; Me4Si) 17.6, 20.9, 27.0, 54.4, 56.1, 104.4,
104.5, 104.6, 106.2, 118.2, 119.0, 119.9, 122.6, 124.1, 127.7, 137.4,
141.2, 147.3, 149.2, 149.5, 150.7 and 152.9; m/z (FAB) 404.1862
(M+ − Br. C25H26NO4 requires 404.1862).
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