Synthesis and antitumor activities of phenanthrene-based alkaloids

Article abstract of DOI:10.3390/molecules14125042

A series of phenanthrene-based tylophorine derivatives (PBTs) were synthesized and their cytotoxic activities against the H460 human large-cell lung carcinoma cell line were evaluated. Among these compounds, N-(3-hydroxy-2,6,7-tri-methoxyphenanthr-9-ylmet

Full text of DOI:10.3390/molecules14125042

Molecules 2009, 14, 5042-5053; doi:10.3390/molecules14125042
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Antitumor Activities of Phenanthrene-Based
Alkaloids
Songtao Li 1,†, Li Han 1,†, Liang Sun 1, Dan Zheng 1, Jiang Liu 1, Yingbo Fu 1, Xueshi Huang 1,*
and Zhanyou Wang 2,*
1
Department of Natural Products Chemistry, Pharmaceutical School, China Medical University,
Shenyang 110001, China
2
Laboratory of Cell Engineering and Cell Therapy, College of Basic Medical Science, China
Medical University, Shenyang 110001, China
†
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-Mails: xshuang@mail.cmu.edu.cn
(X.H.); zhanyouw@hotmail.com (Z.W.).
Received: 26 October 2009; in revised form: 25 November 2009 / Accepted: 2 December 2009 /
Published: 7 December 2009
Abstract: A series of phenanthrene-based tylophorine derivatives (PBTs) were
synthesized and their cytotoxic activities against the H460 human large-cell lung
carcinoma cell line were evaluated. Among these compounds, N-(3-hydroxy-2,6,7-tri-
methoxyphenanthr-9-ylmethyl)-L-prolinol (5a), and N-(3-hydroxy-2,6,7-trimethoxy-
phenanthr-9-ylmethyl)-L-valinol (9) exhibited good activities, with IC50 values of 11.6 and
6.1 μM, respectively.
Keywords: PBTs; synthesis; cytotoxicity
1. Introduction
Phenanthroindolizidine alkaloids have been of considerable interest as anticancer agents because of
their exceptionally potent antitumor activity [1]. Based on recent studies, the molecular target of
phenanthroindolizidine analogues may be novel and different from the targets of known anticancer
drugs [2]. Over the past decades, the synthesis and biological activities of varied 2,3,6,7-functionalized

Molecules 2009, 14
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phenanthroindolizidine alkaloids have been reported. The substituents mainly include functionalities
such as hydroxyl, methoxyl, and hydrogen [3–5]. The structure activity relationships of
phenanthroindolizidine alkaloids were summarized by Gao et al. in 2007 [3]. The degree of
cytotoxicity was dependent on the type and pattern of substitution on the phenanthrene ring. In 2006,
Wei et al. [6] found that phenanthrene-based tylophorine derivatives (PBTs), which were
phenanthroindolizidine derivatives resulting from opening of the indolizidine ring, exhibited
significant cytotoxic activity. Further work from the same group resulted in the synthesis of a series of
PBTs with different substituents at C-9, which indicated that a five- or six-carbon distance between the
nitrogen and a terminal polar substituent in the C-9 chain is quite favorable for cytotoxic activity [7].
They also reported that antitumor activities of PBTs were induced by inhibition of the activation of
Akt and NF-κB signaling pathway in tumor cells [8,9].
As part of our studies on antitumor agents derived from natural products, we have designed and
synthesized a series of PBT analogues to investigate the effects of oxygenic functional groups on the
phenanthrene ring and C-9 side chain on cytotoxic activity. The present paper reports the synthesis of
these PBTs (Scheme 1) and evaluation of their cytotoxic activities against the H460 human large-cell
lung carcinoma cell line in vitro (Table 1).
Scheme 1. The synthetic routes to PBTs.
OMe
OMe
RO
RO
OMe
OMe
RO
MeO
A
B
N
+
COOH
COOH
COOMe
CHO
O
MeO
MeO
OMe
OMe
1a: R = H
1b: R = Me
2a: R = Ac, 86%.
2b: R = Me, 90%.
3a: R = Ac, 95%.
3b: R = Me, 96%.
OMe
OMe
RO
RO
C
D
N
N
COOMe
CH2OH
O
MeO
MeO
OMe
OMe
4a: R = Ac, 85%.
4b: R = Me, 84%.
5a: R = H, 90%.
5b: R = Me, 93%.
OMe
OMe
AcO
OMe
AcO
HO
H
OMe
OMe
H
N
H
N
G
N
OH
F
E
H
2a
O
O
O
O
O
MeO
MeO
MeO
OMe
8, 83%.
OMe
OMe
6, 91%.
7, 86%.
OMe
OMe
I
10: R = OAc, 27%.
11: R = OSO3H, 17%.
12: R = Cl, 85%.
HO
HO
I
H
N
OH
N
CH2R
J
MeO
MeO
OMe
9, 78%.
OMe
5a: R = OH
Reagents and Conditions: A). Et3N/ Ac2O, 140℃, 7 h; B). (COCl)2, DMF/ THF, r.t., 4 h;
Proline methyl ester hydrochlorate, Py/ CH2Cl2, r.t, overnight; C). FeCl3/ CH2Cl2, r.t., 2 h;
D). LAH/ THF, 100℃, 2 h; E). (COCl)2, DMF/ THF, r.t., 4 h; Valine methyl ester
hydrochlorate, Py/ CH2Cl2, r.t., overnight; F). FeCl3/ CH2Cl2, r.t. 2 h; G). LAH/ THF, 100℃,
2 h; H). NaBH4,I2/ THF, 100℃, 4 h; I). con-H2SO4/ HOAc, -15℃, 20 min, 35℃, 0.5 h; J).
SOCl2/ CH2Cl2, 45℃, 1.5 h.

Molecules 2009, 14
Table 1. The structures of 5a, 5b, 8-12 and their cytotoxicities against H460 cell line.
5044
OMe
R1O
R2
MeO
OMe
Compound
R1
H
R2
IC50a (μM)
N
5a
11.6
CH2OH
5b
8
OMe
H
53.8
68.1
6.1
N
CH2OH
OH
H
N
O
H
N
9
H
OH
10
11
12
H
46.8
53.4
62.9
N
CH2OAc
H
N
CH2OSO3H
CH2Cl
N
H
Adriamycin used as positive control, IC50 = 1.72 μM.
2. Results and Discussion
2.1. Chemical synthesis
The synthetic routes to the PBTs are depicted in Scheme 1. Perkin condensation [10] involving
4-hydroxy-3-methoxy- or 3,4-dimethoxybenzaldehyde and 3,4-dimethoxyphenylacetic acid in the
presence of Ac2O and Et3N afforded the cinnamic acid derivatives 2a (R = Ac) or 2b (R = Me)
[11–14]. By treatment with oxalyl chloride [15], the acids 2a or 2b gave an acid chloride intermediate
which could be converted to the corresponding aromatic amides 3a (R = Ac) or 3b (R = Me) [10] by
using L-proline methyl ester hydrochloride. Through oxidation-coupling with ferric trichloride [11] as
the catalyst, the amides were efficiently cyclized to deliver 9-amido-substituted phenanthrenes 4a
(R = Ac) and 4b (R = Me) [10,16], respectively. Finally, the two amides were reduced with LiAlH4
[17] to give 5a (R = OH) and 5b (R = Me) [18,19]. By treatment with concentrated H2SO4/HOAc [20]

Molecules 2009, 14
5045
or SOCl2/CH2Cl2 [21], 5a was converted to 10 and 11, or 12. Compound 7 was synthesized from 2a
and L-valine methyl ester hydrochloride following the same synthetic route as described for 4a.
In our synthetic route, the key step is the reduction of the aromatic amides with aluminum and
boron hydrides. In the reduction process we tried LiAlH4/THF [17], NaBH4-MeSO3H/DMSO [22] and
NaBH4-I2/THF [23] systems to reduce compound 7. First, reduction with the NaBH4-MeSO3H/DMSO
system did not afford the target product, but rather a series of small amounts of unidentified products.
Then, by using LiAlH4 as the catalyst, the 3-acetoxyl on the phenanthrene ring and the ester group in
the amido side chain of 7 were reduced to hydroxyl and hydroxymethyl groups, respectively, as shown
for compound 8, but the carbonyl linkage was not reduced. Finally, under the catalysis of NaBH4-
I2/THF system, 8 was successfully reduced to 9 in a satisfactory yield of 78%. However, compounds
4a and 4b were completely reduced using the LiAlH4/THF system in one step to give 5a and 5b in
consistently high yields of 90% and 93%, respectively.
2.2. Biological activity
The synthesized PBTs were screened for in vitro cytotoxic activity against the H460 human large-
cell lung carcinoma cell line with an MTT assay procedure [24]. Adriamycin was used as the reference
compound. Table 1 summarizes the structures of 5a, 5b, 8-12 and their cytotoxicities (IC50) against the
H460 cell line. All of them exhibited diminished cytotoxic activities to some extent, in comparison to
their parent natural products (e.g., tylophorine, IC50 0.5~1.7 μM) [25].
The results indicate that cytotoxicities of PBTs were influenced by the presence of oxygenic
functional substituents on the phenanthrene, and amino acid side chains at the C-9 position of
phenanthrene and the linkage between the nitrogen and the phenanthrene. Replacing the 3-methoxyl
on the phenanthrene skeleton with a hydroxyl increased the cytotoxic activity, as shown in the
comparison of 5b (3-methoxyl, IC50 53.8 μM) and 5a (3-hydroxyl, IC50 11.6 μM). Thus, a hydroxyl at
the phenanthrene C-3 is quite favorable for cytotoxic activity. In the prolinol side chain, when the
terminal hydroxyl was converted to an acetate ester, sulfate ester and chloride, the cytotoxic activities
were significantly diminished, it may relate to the removal of the terminal hydrogen bond effect on the
assumed biological target [7]. Conserving the C-3 hydroxyl substitution on the phenanthrene skeleton
of 5a and opening the pentacyclic prolinol resulted in a more active compound 9 (IC50 6.1μM). This
result indicates that changing the cyclic side chain to an acyclic one may increase the flexibility of the
C-9 side chain, which favors attaining an optimal conformation for binding to an assumed biological
target. The amide derivative 8 exhibited very low activity (IC50 68.1μM), in agreement with the report
of Wei et al. [6].
3. Experimental
3.1. General
Melting points of the synthesized compounds were determined on a Digital Melting Point Apparatus
XT4A and are uncorrected. IR spectra were recorded with a Perkin-Elmer model 298 spectrometer
(KBr). 1D NMR spectra were recorded on Bruker ARX-300 or Bruker AV-600 spectrometers. ESIMS
were recorded with a Finnigan LCQ mass spectrometer and EIMS were measured by an Agilent/HP

Molecules 2009, 14
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HP5973 and 6890 GC/MSD. Column chromatography was carried out on silica gel (200-300 mesh,
Qingdao Marine Chemical Ltd., Qingdao, China). The MTT assay was recorded on a microplate reader
(KHB ST-360, SH Kehua Laboratory System Co., Ltd., Shanghai, China). Thin-layer chromatography
(TLC) was performed on TLC silica gel 60 F254 plates (0.5 mm, Merck). Chemicals from Sinopharm
Chemical Reagent Co. were used without further purification.
3.2. Preparation of PBT derivatives
3.2.1. Synthesis and spectroscopic data of 4-acetoxy-α-(3,4-dimethoxyphenyl)-3-methoxycinnamic
acid (2a) and 3,4-dimethoxy-α-(3,4-dimethoxyphenyl)cinnamic acid (2b)
A mixture of 4-hydroxy-3-methoxybenzaldehyde (1a, 0.15 g, 1 mmol), 3,4-dimethoxyphenylacetic
acid (0.19 g, 1 mmol), acetic anhydride (2 mL) and triethylamine (0.3 mL) was heated to reflux at
140 °C for 7 h using a CaCl2 tube. Then the mixture was cooled to room temperature, diluted with
water (10 mL) and refluxed at 120 °C for 30 min. The cooled mixture was extracted with EtOAc
(3 × 50 mL). The combined organic extracts were washed with saturated brine (150 mL), dried over
anhydrous Na2SO4, filtered, concentrated in vacuo and the residue recrystallized from methanol to
afford 2a (0.32 g, 86%) as pale-yellow crystals. m.p. 166-168 °C; 1H-NMR (300 MHz, CDCl3) δ: 2.28
(s, 3H, CH3CO), 3.47 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 6.67 (d, 1H, J = 1.5 Hz,
Ar-H), 6.79 (d, 1H, J = 1.5 Hz, Ar-H), 6.83 (dd, 1H, J = 8.2, 1.5 Hz, Ar-H), 6.84 (dd, 1H, J = 8.1,
1.5 Hz, Ar-H), 6.91 (d, 1H, J = 8.1 Hz, Ar-H), 6.92 (d, 1H, J = 8.2 Hz, Ar-H), 7.87 (s, 1H, C=CH); IR
(KBr) cm-1 ν: 3421, 3062, 2964, 2839, 1764, 1674, 1619, 1516; EIMS m/z: 372 [M]+ (C20H20O7), 330
(100%) [M-acyl]+. Compound 2b (0.31 g, 90%) was synthesized as a deep yellow solid from
3,4-dimethoxy-benzaldehyde (1b, 0.17 g, 1 mmol) and 3,4-dimethoxyphenylacetic acid (0.19 g,
1
1 mmol) in the same synthetic route as that for 2a. m.p. 217-219 °C; H-NMR (300 MHz, CDCl3) δ:
3.47 (s, 3H, OCH3), 3.81 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.89 (s, 3H, OCH3), 6.55 (d, 1H,
J = 1.8 Hz, Ar-H), 6.70 (d, 1H, J = 1.8 Hz, Ar-H), 6.72 (d, 1H, J = 8.5 Hz, Ar-H), 6.84 (dd, 1H, J = 8.2,
1.8 Hz, Ar-H), 6.85 (dd, 1H, J = 8.5, 1.8 Hz, Ar-H), 6.92 (d, 1H, J = 8.2 Hz, Ar-H), 7.86 (s, 1H,
C=CH); IR (KBr) cm-1 ν: 3448, 3043, 2938, 2835, 1668, 1593, 1510; ESI MS m/z: [M-H]- 343
(C19H20O6).
3.2.2. Synthesis and spectroscopic data of methyl N-[4-acetoxy-α-(3,4-dimethoxyphenyl)-3-methoxy)-
cinnamyl]pyrrolidine-2-carboxylate (3a) and methyl N-[3,4-dimethoxy-α-(3,4-dimethoxyphenyl)-
cinnamyl]pyrrolidine-2-carboxylate (3b)
To a stirred solution of 2a (0.74 g, 2 mmol) and DMF (0.1 mL) in anhydrous THF (10 mL), oxalyl
chloride (2 mL, 22.4 mmol) in anhydrous THF (2 mL) was added dropwise. After the addition was
complete, the mixture was stirred at room temperature for 4 h. Excess oxalyl chloride was removed
under reduced pressure. The yellow residue was dissolved in anhydrous CH2Cl2 (20 mL), to which was
added dropwise a solution of L-proline methyl ester hydrochloride (0.35 g, 2.1 mmol) and pyridine
(1 mL, 12.4 mmol) in anhydrous CH2Cl2 (4 mL) with stirring. The mixture was stirred overnight at
room temperature. Then 1 N HCl (15 mL) was added to the reaction system with vigorous stirring to
quench the reaction. The organic layer was seperated and the aqueous layer was extracted with CH2Cl2

Molecules 2009, 14
5047
(2 × 30 mL). The combined organic extracts were washed with saturated brine (90 mL) and dried over
anhydrous Na2SO4. After filtration and evaporation in vacuo, the residue was purified by column
chromatography on silica gel (hexane/EtOAc, 1:1.25 v/v) to afford 3a (0.92 g, 95%) as a colorless
solid; m.p. 53-55 °C; 1H-NMR (300 MHz, CDCl3) δ: 1.81-1.94 (m, 3H overlapped, 1.5×CH2), 2.23 (m,
1H, 0.5×CH2), 2.26 (s, 3H, CH3CO), 3.30 (m, 2H, CH2N), 3.53 (s, 3H, OCH3), 3.73 (s, 3H, OCH3),
3.76 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 4.55 (m, 1H, CHN), 6.76-6.92 (m, 7H overlapped, 7×Ar-H);
IR (KBr) cm-1 ν: 3041, 2954, 2836, 1764, 1745, 1633, 1600, 1513; EIMS m/z: 483 [M]+ (C26H29NO8),
441 [M-acyl]+, 424 [M-COOMe]+, 327 [M-CO-C6H10NO2]+, 285 (100%) [M-CO-C6H10NO2 -acyl]+.
Compound 3b (1.27 g, 96%) was synthesized as a light yellow solid from 2b (1 g, 2.9 mmol) and
L-proline methyl ester hydrochloride (0.49 g, 3.0 mmol) by the same synthetic route as that for 3a; m.p.
1
44-46 °C; H-NMR (300 MHz, CDCl3) δ: 1.81-1.98 (m, 3H overlapped, 1.5×CH2), 2.24 (m, 1H,
0.5×CH2), 3.34 (m, 2H, CH2N), 3.57 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.88 (s,
3H, OCH3), 4.56 (m, 1H, CHN), 6.70-6.95 (m, 7H overlapped, 7×Ar-H); IR (KBr) cm-1 ν: 3047, 2953,
2836, 1743, 1632, 1600, 1514; EIMS m/z: 455 [M]+ (C25H29NO7), 327 [M-C6H10NO2]+, 299 (100%)
[M-CO-C6H10NO2]+.
3.2.3. Synthesis and spectroscopic data of methyl N-(3-acetoxy-2,6,7-trimethoxyphenanthr-9-
ylcarbonyl)pyrrolidine-2-carboxylate (4a) and methyl N-(2,3,6,7-tetramethoxyphenanthr-9-
ylcarbonyl)-pyrrolidine-2-carboxylate (4b)
To a solution of 3a (0.48 g, 1 mmol) in anhydrous CH2Cl2 (15 mL) was added anhydrous FeCl3
(0.68 g, 4.3 mmol), and the mixture was stirred at room temperature for 2 h under nitrogen. Then 1 N
HCl (1 mL) diluted with saturated brine (10 mL) was added to the stirred reaction system. The organic
layer was seperated and the aqueous layer was extracted with CH2Cl2 (2 × 30 mL). The combined
organic extracts were washed with saturated brine (80 mL) and dried over anhydrous Na2SO4. After
filtration and evaporation, the residue was purified by column chromatography on silica gel
(hexane/EtOAc, 1:1.5 v/v) to afford 4a (0.41 g, 85%) as an off-white solid; m.p. 229-231 °C; 1H-NMR
(600 MHz, CDCl3) δ: 1.88 (m, 1H, 0.5×CH2), 1.95 (m, 1H, 0.5×CH2), 2.09 (m, 1H, 0.5×CH2), 2.37 (m,
1H, 0.5×CH2), 2.43 (s, 3H, CH3CO), 3.22 (m, 1H, 0.5×CH2N), 3.36 (m, 1H, 0.5×CH2N), 3.84 (s, 3H,
CH3O), 3.97 (s, 3H, CH3O), 4.00 (s, 3H, CH3O), 4.01 (s, 3H, CH3O), 4.84 (dd, 1H, J = 8.4, 4.8 Hz,
CHN), 7.29 (s, 1H, Ar-H), 7.59 (s, 1H, Ar-H), 7.70 (s, 1H, Ar-H), 7.77 (s, 1H, Ar-H), 8.14 (s, 1H, Ar-
H); IR (KBr) cm-1 ν: 3083, 2953, 2836, 1763, 1743, 1631, 1508; ESI MS m/z: [M+H]+
482 (C26H27NO8). Compound 4b (1.33 g, 84%) was synthesized as a light yellow solid from 3b (1.6 g,
3.5 mmol) and anhydrous FeCl3 (2.27 g, 14 mmol) by a similar synthetic route as that of 4a; m.p. 200-
1
202 °C; H-NMR (300 MHz, CDCl3) δ: 1.77-1.98 (m, 2H overlapped, CH2), 2.06 (m, 1H, 0.5×CH2),
2.32 (m, 1H, 0.5×CH2), 3.22 (m, 1H, 0.5×CH2N), 3.36 (m, 1H, 0.5×CH2N), 3.81 (s, 3H, CH3O), 3.98
(s, 3H, CH3O), 4.06 (s, 3H, CH3O), 4.09 (s, 6H, 2×CH3O), 4.81 (m, 1H, CHN), 7.14 (s, 1H, Ar-H),
7.52(s, 1H, Ar-H), 7.64 (s, 1H, Ar-H), 7.73 (s, 1H, Ar-H), 7.74 (s, 1H, Ar-H); IR (KBr) cm-1 ν: 3077,
2952, 2836, 1741, 1628, 1509; EIMS m/z: 453 [M]+ (C25H27NO7), 325 (100%) [M-C6H10NO2]+.

Molecules 2009, 14
5048
3.2.4. Synthesis and spectroscopic data of N-(3-hydroxy-2,6,7-trimethoxyphenanthr-9-ylmethyl)-L-
prolinol (5a) and N-(2,3,6,7-tetramethoxyphenanthr-9-ylmethyl)-L-prolinol (5b)
To a stirred, ice-salt bath cooled solution of 4a (3.1 g, 6.4 mmol) in anhydrous THF (100 mL) was
added LiAlH4 (1.3 g, 39 mmol) in one portion. After being stirred for 30 min kept at 0 °C, the mixture
was heated to reflux at 100 °C for 2 h using a CaCl2 tube. To the cooled mixture were added ice and
saturated NaHCO3 solution (100 mL). After stirring for 10 min, the THF layer was separated and the
aqueous layer was extracted with THF (2 × 50 mL). The combined organic extracts were washed with
saturated brine (200 mL) and dried over anhydrous Na2SO4. After filtration and evaporation, the
residue was purified by column chromatography on silica gel (CH2Cl2/MeOH, 20:1 v/v) to afford 5a
1
(2.3 g, 90%) as an off-white solid. m.p. 178-180 °C; H-NMR (600 MHz，CDCl3) δ: 1.70 (m, 1H,
0.5×CH2), 1.74 (m, 1H, 0.5×CH2), 1.87 (m, 1H, 0.5×CH2), 2.02 (m, 1H, 0.5×CH2), 2.46 (m, 1H,
0.5×CH2N), 2.95 (m, 1H, 0.5×CH2N), 2.97 (m, 1H, CHN), 3.52 (d, 1H, J = 10.8 Hz, 0.5×CH2O), 3.74
(d, 1H, J = 12.6 Hz, 0.5×ArCH2N), 3.79 (d, 1H, J = 10.8 Hz, 0.5×CH2O), 4.03 (s, 3H, CH3O), 4.08 (s,
6H, 2×CH3O), 4.47 (d, 1H, J = 12.6 Hz, 0.5×ArCH2N), 7.18 (s, 1H, Ar-H), 7.50 (s, 1H, Ar-H), 7.57 (s,
1H, Ar-H), 7.82 (s, 1H, Ar-H), 7.95 (s, 1H, Ar-H); IR (KBr) cm-1 ν: 3388, 3050, 2953, 2839, 1617,
1513; ESI MS m/z: 398 [M+H]+ (C23H27NO5). Compound 5b (0.84 g, 93%) was synthesized as a white
solid from 4b (1 g, 2.2 mmol) and LiAlH4 (0.7 g, 21 mmol) by the same synthetic route as that
described for 5a; m.p. 217-218 oC; 1H-NMR (300 MHz, CDCl3) δ: 1.57-1.67 (m, 2H overlapped, CH2),
1.79 (m, 1H, 0.5×CH2), 1.91 (m, 1H, 0.5×CH2), 2.38 (m, 1H, 0.5×CH2N), 2.80 (m, 1H, 0.5×CH2N),
2.87 (m, 1H, CHN), 3.42 (dd, 1H, J = 10.5, 2.4 Hz, 0.5×CH2O), 3.65 (d, 1H, J = 12.6 Hz,
0.5×ArCH2N), 3.68 (dd, 1H, J = 10.5, 3.6 Hz, 0.5×CH2O), 3.96 (s, 3H, CH3O), 4.00 (s, 3H, CH3O),
4.02 (s, 3H, CH3O), 4.04 (s, 3H, CH3O), 4.39 (d, 1H, J = 12.6 Hz, 0.5×ArCH2N), 7.12 (s, 1H, Ar-H),
7.44 (s, 1H, Ar-H), 7.52 (s, 1H, Ar-H), 7.70 (s, 1H, Ar-H), 7.74 (s, 1H, Ar-H); IR (KBr) cm-1 ν: 3423,
3050, 2958, 2836, 1617, 1510; ESI MS m/z: 412 [M+H]+ (C24H29NO5).
3.2.5. Synthesis and spectroscopic data of N-[4-acetoxy-α-(3,4-dimethoxyphenyl)-3-methoxy-
cinnamyl]-L-valine methyl ester (6)
Compound 6 (1.90 g, 91%) was synthesized as a colorless solid from 2a (1.6 g, 4.3 mmol) and
L-valine methyl ester hydrochloride (0.75 g, 4.5 mmol) by a similar synthetic route as that for 3a;
m.p. 46-48 °C; 1H-NMR (300 MHz, CDCl3) δ: 0.76 (d, 3H, J = 6.9 Hz, CH3), 0.91 (d, 3H, J = 6.8 Hz,
CH3), 2.13 (m, 1H, CH), 2.27 (s, 3H, CH3CO), 3.48 (s, 3H, OCH3), 3.71 (s, 3H, OCH3), 3.84 (s, 3H,
OCH3), 3.93 (s, 3H, OCH3), 4.65 (brdd, 1H, J = 8.7, 4.9, CHN), 6.10 (d, 1H, J = 8.7 Hz, NH), 6.62 (d,
1H, J = 1.7 Hz, Ar-H), 6.75 (dd, 1H, J = 8.1, 1.7 Hz, Ar-H), 6.81 (d, 1H, J = 1.7 Hz, Ar-H), 6.85 (d,
1H, J = 8.2 Hz, Ar-H), 6.88 (dd, 1H, J = 8.2, 1.7 Hz, Ar-H), 6.98 (d, 1H, J = 8.1 Hz, Ar-H), 7.77 (s,
1H, C=CH); IR (KBr) cm-1 ν: 3420, 3050, 2961, 2836, 1767, 1740, 1670, 1616, 1513; EIMS m/z: 485
[M]+ (C26H31NO8), 443 (100%) [M-acyl]+, 328[M-CO-C6H12NO2]+, 285[M-acyl-CO-C6H12NO2]+.

Molecules 2009, 14
5049
3.2.6. Synthesis and spectroscopic data of N-(3-acetoxy-2,6,7-trimethoxyphenanthr-9-ylcarbonyl)-L-
valine methyl ester (7)
Compound 7 (335 mg, 86%) was synthesized as an off-white solid from 6 (390 mg, 0.80 mmol) and
anhydrous FeCl3 (0.53 g, 3.24 mmol) using the same synthetic route as that for 4a; m.p. 205-206 °C;
1H-NMR (300 MHz, CDCl3) δ: 1.02 (d, 3H, J = 6.9 Hz, CH3), 1.12 (d, 3H, J = 6.8 Hz, CH3), 2.36 (m,
1H, CH), 2.43 (s, 3H, CH3CO), 3.83 (s, 3H, OCH3), 3.98 (s, 3H, OCH3), 4.01 (s, 3H, OCH3), 4.09 (s,
3H, OCH3), 4.93 (brdd, 1H, J = 8.8 Hz, 4.9Hz, CHN), 6.64 (d, 1H, J = 8.8 Hz, NH), 7.29 (s, 1H,
Ar-H), 7.73 (s, 1H, Ar-H), 7.76 (s,1H, Ar-H), 7.79 (s, 1H, Ar-H), 8.11(s, 1H, Ar-H); IR (KBr) cm-1 ν:
3288, 3050, 2961, 2834, 1760, 1742, 1635, 1598, 1508; ESI MS m/z: 482[M-H]- (C26H28NO8).
3.2.7. Synthesis and spectroscopic data of N-(3-hydroxy-2,6,7-trimethoxyphenanthr-9-ylcarbonyl)-L-
valinol (8)
A 100 mL dry three-necked flask was charged with a stirred suspension of LiAlH4 (61 mg,
1.6 mmol) in anhydrous THF (15 mL) kept at −10~0 °C for 10 min. Compound 7 (150 mg, 0.31 mmol)
dissolved in anhydrous THF (15 mL) was added dropwise to the suspension with stirring at
−10~0 °C. After the addition was complete, stirring was continued for 10 min, then the warmed
reaction system was refluxed at 100 °C for 2 h using a CaCl2 tube. To the cooled mixture were added
ice and saturated NaHCO3 solution (30 mL). After being stirred for 10 min, the THF layer was
separated and the aqueous layer was extracted with CH2Cl2 (2 × 30 mL). The combined organic
extracts were washed with saturated brine (90 mL), dried over anhydrous Na2SO4, filtered and
concentrated in vacuo. The residue was purified by column chromatography on silica gel
1
(CH2Cl2/MeOH, 30:1 v/v) to afford 8 (106 mg, 83%) as a white solid; m.p.182-183 °C; H-NMR
(300 MHz, DMSO) δ: 0.94 (d, 3H, J = 6.8 Hz, CH3), 0.99 (d, 3H, J = 6.8 Hz, CH3), 1.98 (m, 1H, CH),
3.56 (m, 2H, CH2O), 3.81 (s, 3H, OCH3), 3.92(s, 3H, OCH3), 3.96(m, 1H, CHN), 3.98(s, 3H, OCH3),
7.43 (s, 1H, Ar-H), 7.69 (s, 1H, Ar-H), 7.70 (s, 1H, Ar-H), 7.85 (s, 1H, Ar-H), 7.98 (s, 1H, Ar-H),
8.12(d, 1H, J = 9.15 Hz, NH); IR (KBr) cm-1 ν: 3296, 3050, 2957, 2835, 1620, 1593, 1508; ESI MS
m/z: 412 [M-H]- C23H26NO6 .
3.2.8. Synthesis and spectroscopic data of N-(3-hydroxy-2,6,7-trimethoxyphenanthr-9-ylmethyl)-L-
valinol (9)
Compound 8 (41 mg, 0.1 mmol) dissolved in anhydrous THF (5 mL) was added dropwise to a
stirred solution of NaBH4 (20 mg, 0.5 mmol) in anhydrous THF (20 mL) kept at −5~0 °C. Then I2
(59 mg, 0.23 mmol) dissolved in anhydrous THF (7 mL) was added dropwise to the stirred reaction
system at −5~0 °C over 2 h. After the addition was complete, the mixture was warmed to room
temperature and heated to reflux at 100 °C for 4 h. Then to the cooled mixture, 1 N HCl (8 mL) was
added dropwise with stirring to quench the excess hydride. After the gas evolution ceased, saturated
NaHCO3 solution (8 mL) was added dropwise to neutralize the excess HCl. The THF layer was
separated and dried over anhydrous Na2SO4. After filtration, a solution of BF3·Et2O (2 mL) in THF
(5 mL) was added dropwise to the filtrate, then saturated NaHCO3 solution (10 mL) was added to
dissociate the target amine. The THF layer was separated, and the aqueous layer was extracted with

Molecules 2009, 14
5050
THF (2 × 30 mL). The combined extracts were washed with saturated brine (100 mL) and dried over
anhydrous Na2SO4. After filtration and evaporation in vacuo, the residue was purified by column
chromatography on silica gel (CH2Cl2/MeOH, 15:1 v/v) to afford 9 (31 mg, 78%) as an off-white
1
solid; m.p. 224-225 °C; H-NMR (300 MHz, DMSO) δ: 0.91 (d, 3H, J = 6.7 Hz, CH3), 0.96 (d, 3H,
J = 6.7 Hz, CH3), 2.15 (m, 1H, CH), 2.98 (m, 1H, CHN), 3.60 (m, 1H, 0.5×OCH2), 3.73 (m, 1H,
0.5×OCH2), 3.92 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 3.98 (s, 3H, OCH3), 4.45 (m, 2H, ArCH2N), 7.33
(s, 1H, Ar-H), 7.59 (s, 1H, Ar-H), 7.68 (s, 1H, Ar-H), 7.86 (s, 1H, Ar-H), 7.97(s, 1H, Ar-H); IR (KBr)
cm-1 ν: 3423, 3050, 2927, 2836, 1618, 1508; ESI MS m/z: [M+H]+ 400 (C23H29NO5).
3.2.9. Synthesis and spectroscopic data of [N-(3-hydroxy-2,6,7-trimethoxyphenanthr-9-ylmethyl)-
pyrrolidin-2-yl]methyl acetate (10) and [N-(3-hydroxy-2,6,7-trimethoxyphenanthr-9-ylmethyl)pyrro-
lidin-2-yl]methyl hydrogen sulfate (11)
To a dry 50 mL three-necked flask charged with 5a (1 g, 2.52 mmol) was added glacial acetic acid
(5 mL) at −15 °C. The mixture was stirred for 5 min. Then frozen concentrated sulfuric acid (10 mL)
was added dropwise to the stirred system at −15 °C. After the addition was complete, stirring was
continued for 20 min. Then the solution was warmed to room temperature and gently heated to 35 °C.
Thirty min later, the reaction mixture was cooled to room temperature, poured into water (100 mL) and
adjusted to pH 10 with 1N KOH solution. The resulting mixture was extracted with EtOAc
(3 × 80 mL). The combined extracts were washed with saturated brine (250 mL), dried over anhydrous
Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography on
silica gel (CH2Cl2/MeOH, 15:1 v/v) to afford 10 and 11. Compound 10 (0.3 g, 27%), a white solid;
1
m.p. 136-137 °C; H-NMR (600 MHz, CDCl3) δ: 1.66 (m, 3H overlapped, 1.5×CH2), 2.01 (m, 1H,
0.5×CH2), 2.06 (s, 3H, CH3CO), 2.35 (m, 1H, 0.5×CH2N), 2.84 (m, 1H, 0.5×CH2N), 2.96 (m, 1H,
CHN), 3.68 (d, 1H, J = 13.2 Hz, 0.5×ArCH2N), 4.03 (s, 3H, OCH3), 4.05 (s, 3H, OCH3), 4.09 (s, 3H,
OCH3), 4.13 (m, 1H, 0.5×CH2O), 4.29 (m, 1H, 0.5×CH2O), 4.61 (d, 1H, J = 13.2 Hz, 0.5×ArCH2N),
7.18 (s, 1H, Ar-H), 7.49 (s, 1H, Ar-H), 7.81 (s, 1H, Ar-H), 7.84 (s, 1H, Ar-H), 7.96 (s, 1H, Ar-H); IR
(KBr) cm-1 ν: 3430, 3050, 2953, 2834, 1736, 1618, 1509; ESI MS m/z: 438 [M-H]-, 440 [M+H]+
1
(C25H29NO6). Compound 11 (0.2 g, 17%), a white solid; m.p. 216-218 °C; H-NMR (600 MHz,
DMSO) δ: 1.78 (m, 1H, 0.5×CH2), 1.80 (m, 1H, 0.5×CH2), 2.02 (m, 1H, 0.5×CH2), 2.23 (m, 1H,
0.5×CH2), 3.10 (m, 1H, 0.5×CH2N), 3.35 (m, 1H, 0.5×CH2N), 3.94 (s, 3H, OCH3), 4.00 (s, 3H,
OCH3), 4.03 (s, 3H, OCH3), 4.14 (m, 2H, CH2O), 4.34 (d, 1H, J = 13.2 Hz, 0.5×ArCH2N), 4.57 (m,
1H, CHN), 5.33 (d, 1H, J = 13.2 Hz, 0.5×ArCH2N), 7.35 (s, 1H, Ar-H), 7.56 (s, 1H, Ar-H), 7.82 (s,
1H, Ar-H), 7.91 (s, 1H, Ar-H), 8.01 (s, 1H, Ar-H); IR (KBr) cm-1 ν: 3423, 3050, 2924, 2850, 1624,
1514; ESI MS m/z: 476 [M-H]-, 478 [M+H]+ (C23H27NO8S).
3.2.10. Synthesis and spectroscopic data of N-(3-hydroxy-2,6,7-trimethoxyphenanthr-9-ylmethyl)-2-
chloromethylpyrrolidine (12)
A 250 mL dry three-necked flask fitted with a dropping funnel and a condenser was charged with
5a (2 g, 5.04 mmol) and anhydrous CH2Cl2 (70 mL). The condenser was fitted with a trap to remove
the vapors of hydrogen chloride and sulfur dioxide. Freshly distilled SOCl2 (5 mL) in anhydrous
CH2Cl2 (30 mL) was added dropwise to the stirred system at 0 °C. After the addition was complete,

Molecules 2009, 14
5051
the mixture was warmed to room temperature and heated to reflux at 45 °C for 1.5 h, after which the
reaction system was cooled to room temperature and adjusted to pH 10 with saturated Na2CO3 solution
(100 mL) at 0 °C. The organic layer was separated and the aqueous layer was extracted with CH2Cl2
(2 × 50 mL), the combined extracts were washed with saturated brine (200 mL) and dried over
anhydrous Na2SO4. After filteration and evaporation in vacuo, the residue was purified by column
chromatography on silica gel (PE/EtOAc, 3:1 v/v) to afford 12 (1.78 g, 85%) as an off-white solid; m.p.
1
184-185 °C; H-NMR (300 MHz, CDCl3) δ: 1.67 (m, 2H overlapped, CH2), 1.82 (m, 1H, 0.5×CH2),
2.12 (m, 1H, 0.5×CH2), 2.26 (m, 1H, 0.5×CH2N), 2.41 (m, 1H, 0.5×CH2Cl), 2.72 (m, 1H, 0.5×CH2N),
3.08 (m, 1H, 0.5×CH2Cl), 3.83 (d, 1H, J = 13.2 Hz, 0.5×ArCH2N), 3.97 (d, 1H, J = 13.2 Hz,
0.5×ArCH2N), 3.98 (m, 1H, CHN), 4.03 (s, 3H, OCH3), 4.05 (s, 3H, OCH3), 4.10 (s, 3H, OCH3), 7.17
(s, 1H, Ar-H), 7.44 (s, 1H, Ar-H), 7.82 (s, 1H, Ar-H), 7.86 (s, 1H, Ar-H), 7.96 (s, 1H, Ar-H); IR (KBr)
cm-1 ν: 3442, 3050, 2947, 2832, 1619, 1508; ESI MS m/z: 416 [M+H]+ (C23H26ClNO4).
3.3. Cytotoxic assay
Cytotoxicities were determined by MTT method [24] using human large-cell lung carcinoma cell
line (H460) grown in RPM1-1640 medium plus 10% heat-inactived fetal bovine serum. The assays
were performed in 96-well microtiter plates. Compounds 5a, 5b, 8-12 were dissolved in DMSO and
diluted to six different concentrations (10, 3.3, 1.0, 0.33, 0.10, and 0.033 mM) respectively, and each
solution was ten-fold diluted to six different concentrations using culture medium (1.0, 0.33, 0.10,
0.033, 0.010, and 0.0033 mM), then 10 µL of each solution was added to 90 µL (about 5,000 cells)
culture medium wells. After incubation at 37 °C for 72 hours, 10 µL of MTT (5 mg/mL) was added to
each well and incubated for four hours, and then liquid in the wells was removed. DMSO (150 µL)
was added to each well. The absorbance was recorded on a microplate reader at wavelength of 590 nm,
and the IC50 was defined as 50% reduction of absorbance in the control assay. Compound 5a, 5b, 8-12
showed cytotoxicity against human tumor cell line H460 with IC50 value of 11.6, 53.8, 68.1, 6.1, 46.8,
53.4 and 62.9 μM, respectively. Adriamycin, with an IC50 value of 1.72 μM, was used as
positive control.
4. Conclusions
In conclusion, we have synthesized a series of PBT analogues by a facile route and evaluated their
in vitro cytotoxic activities against human large-cell lung carcinoma cell line (H460). On this basis, the
SAR analysis and potential antitumor activities of these alkaloids are under investigation. Compounds
5a and 9 with a substitution of hydroxyl at position 3 and a hydroxyl at C-9 side chain terminus were
the most effective cytotoxic compounds. These results may be helpful for the design of future PBT
antitumor reagents, and offer potential application in the discovery of antitumor drugs.
Acknowledgements
This work was founded by National Natural Science Foundation of China (Grant No. 30600783).

Molecules 2009, 14
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Sample Availability: Samples of compounds 2–12 are available from the authors.
© 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.
This article is an open-access article distributed under the terms and conditions of the Creative
Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).