Antitumor agents 251: Synthesis, cytotoxic evaluation, and structure-activity relationship studies of phenanthrene-based tylophorine derivatives (PBTs) as a new class of antitumor agents

Article abstract of DOI:10.1016/j.bmc.2006.06.009

Polar phenanthrene-based tylophorine derivatives (PBTs) were designed, synthesized and evaluated as potential antitumor agents. These compounds contain a core phenanthrene structure and can be synthesized efficiently in excellent yield. The newly synthesized PBTs were evaluated for cytotoxic activity against the A549 human cancer cell line. Among them, N-(2,3-methylenedioxy-6-methoxy-phenanthr-9-ylmethyl)-l-2-piperidinemethanol (34) and N-(2,3-methylenedioxy-6-methoxy-phenanthr-9-ylmethyl)-5-aminopentanol (28) showed the highest potency with IC₅₀ values of 0.16 and 0.27 μM, respectively, which are comparable to those of currently used antitumor drugs. A structure-activity relationship (SAR) study was also explored to facilitate the further development of this new compound class.

Full text of DOI:10.1016/j.bmc.2006.06.009

Bioorganic & Medicinal Chemistry 14 (2006) 6560–6569
Antitumor agents 251: Synthesis, cytotoxic evaluation,
and structure–activity relationship studies of phenanthrene-based
tylophorine derivatives (PBTs) as a new class of antitumor agents
Linyi Wei, Arnold Brossi, Ross Kendall, Kenneth F. Bastow,
Susan L. Morris-Natschke, Qian Shi and Kuo-Hsiung Lee*
Natural Products Research Laboratories, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
Received 31 March 2006; revised 2 June 2006; accepted 6 June 2006
Available online 30 June 2006
Abstract—Polar phenanthrene-based tylophorine derivatives (PBTs) were designed, synthesized and evaluated as potential antitu-
mor agents. These compounds contain a core phenanthrene structure and can be synthesized efficiently in excellent yield. The newly
synthesized PBTs were evaluated for cytotoxic activity against the A549 human cancer cell line. Among them, N-(2,3-methylenedi-
oxy-6-methoxy-phenanthr-9-ylmethyl)-L-2-piperidinemethanol (34) and N-(2,3-methylenedioxy-6-methoxy-phenanthr-9-ylmethyl)-
5-aminopentanol (28) showed the highest potency with IC50 values of 0.16 and 0.27 lM, respectively, which are comparable to those
of currently used antitumor drugs. A structure–activity relationship (SAR) study was also explored to facilitate the further devel-
opment of this new compound class.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
OMe
OMe
2
MeO
MeO
Natural products have been the major source of current-
ly available anticancer drugs. According to a review of
New Chemical Entities (NCE) from 1981 to 2002,1
approximately 74% of anticancer drugs were either nat-
ural products, natural product-based, or mimicked them
in one form or another. The phenanthroindolizidine
alkaloid tylophorine (1, Chart 1) and its analogs have
been isolated primarily from the genera Cynanchum,
Pergularia, and Tylophora in the Asclepiadaceae fami-
ly,2–5 but have also been reported from Hypoestes verti-
cillaris (Acanthaceae),6 Cryptocarya phyllostemmon
(Lauraceae),7 as well as Ficus hispida and Ficus septica
(Moraceae).8,9 These compounds, commonly called
tylophora alkaloids, have been targets of synthesis and
modification for their significant cytotoxic activi-
ties.6,10–13 Evaluation of 1 and its analogs in the Nation-
al Cancer Institute’s antitumor screen showed a uniform
and potent growth inhibitory effect (GI50 ffi 10À8 M)
against all 60 cell lines, with notable selectivity toward
1
H
H
N
3
4
14
13
12
10 11
13a
N
MeO
MeO
5
9
6
8
MeO
7
OMe
(-)-R-Tylophorine (1)
Tylocrebrine (2)
Chart 1.
several refractory cell lines, including melanoma and
lung tumor cell lines.14
Comprehensive evaluation of 1’s antitumor activity has
not been reported, and the inhibitory mechanisms on
cell growth are largely unknown. Early mechanistic
studies in 1960s demonstrated that tylophorine alkaloids
irreversibly inhibit protein synthesis at the elongation
stage of the translation cycle.15–19 With the significant
advances in the field of molecular biology in the 1990s,
tylophorine alkaloids were reported to target key meta-
bolic enzymes, including thymidylate synthase (TS)20,21
Keywords: Phenanthrene-based tylophorine derivatives; Antitumor
agents; Cytotoxicity; Tylophora.
*
Corresponding author. Tel.: +1 919 962 0066; fax: +1 919 966
3893; e-mail: khlee@unc.edu
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.06.009

L. Wei et al. / Bioorg. Med. Chem. 14 (2006) 6560–6569
6561
Table 1. SAR of 2,3-methylenedioxy-6-alkyloxy-9-substituted PBT
analogs
Table 1 (continued)
a,b
IC50 (lM)
Compound
R1
R2
O
27
28
CH3
CH3
–CH2NH(CH2)4COOH
–CH2NH(CH2)4CH2OH
1.3
0.27
2
O
3
HOOC
N
29
30
31
32
CH3
CH3
CH3
CH3
5.3
73.8
2.1
R2
9
O
R1O
6
MeOOC
a,b
Compound
R1
R2
IC50
(lM)
N
7a
7b
–CH2C6H5
–CH3
–COOH
–COOH
NA
NA
41.2
41.2
1.6
HOOC
N
10
–CH2C6H5
–CH2C6H5
–CH2C6H5
–CH2C6H5
–CH2C6H5
–CH2C6H5
–CONH(CH2)5COOMe
–CONH(CH2)5COOH
–CH2NH(CH2)5COOH
–CH2NH(CH2)5CH2OH
–CH2NH(CH2)4COOMe
–CH2NH(CH2)4COOH
11
12
13
14
15
HO
N
1.1
0.7
17.0
2.2
HOOC
N
HOOC
N
16
17
–CH2C6H5
–CH2C6H5
42.6
32.1
33
34
CH3
CH3
0.5
O
HO
N
MeOOC
0.16
N
a Etoposide (VP-16) used as positive control, IC50 = 1.4 lM.
b NA, not active.
HOOC
N
18
19
20
–CH2C6H5
–CH2C6H5
–CH2C6H5
4.4
1.8
3.2
and dihydrofolate reductase.22 In addition, they were
also found to induce cell apoptosis.23 In 2004, Gao
et al.24 found that tylophorine derivatives significantly
inhibit activator protein-1, CRE, and NF-jB mediated
transcription. These recent discoveries emphasize that
tylophorine derivatives merit development as a new anti-
tumor drug class distinctly different from current cancer
chemotherapeutic agents.
HO
N
HOOC
N
In the early 1960s, tylocrebrine (2), a positional isomer
of tylophorine, was advanced to clinical trials but failed
due to its significant CNS toxicity, manifested as disori-
entation and ataxia.13 Stærk et al.25 proposed that the
CNS side effects could possibly be minimized by use of
more polar analogs that would be unable to pass the
blood–brain barrier. Our preliminary screening results
showed that polar intermediate compound 15, an amino
acid precursor of tylophorine, exhibited significant cyto-
toxic activity against a human lung cancer cell line with
an IC50 value of 2.2 lM. This discovery led us to explore
a new series of novel phenanthrene-based tylophorine
derivatives (PBTs). Successful application of the Pschorr
cyclization26 and coupling reaction in the presence of
N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydro-
chloride (EDC) enabled efficient and concise access to
more polar PBTs with overall yields greater than 50%.
In comparison to the reported total synthesis of
tylophorine,27 our PBT synthesis avoids the cyclization
step (limiting synthetic factor) and makes the parallel
and large-scale synthesis of PBTs feasible, which could
potentially enhance the development of these
HO
N
21
22
–CH2C6H5
1.3
HOOC
N
–H
39.7
O
HOOC
N
23
24
–H
–H
41.2
39.7
HOOC
N
25
26
CH3
CH3
–CONH(CH2)4COOH
73.3
25.3
–CH2NH(CH2)4COOMe

6562
L. Wei et al. / Bioorg. Med. Chem. 14 (2006) 6560–6569
OR1
OR1
NH2
COOH
OR1
NO2
R2O
R2O
R2O
c
b
a
+
CHO
NO2
OR3
COOH
COOH
4a: R3= -CH2Ph
4b: R3=CH3
3a: R1,R2 = -CH2-
3b: R1=CH3, R2=CH3
R3O
R3O
-
5a: R1,R2 = -CH2 ; R3=-CH2Ph
5b: R1=CH3, R2=CH3; R3=CH3
5c: R1,R2 = -CH2 ; R3=CH3
6a, b, c
-
OR1
OR1
R2O
OR1
2
R2O
R2O
R3O
1
3
4
10
9
d
e
N-alkyl-R4
N-alkyl-R4
5
COOH
6
8
O
R3O
R3O
7
7a, b, c
9
8
f
R3 = -CH2C6H5
R4 = -COOCH3
R3 = -H
g
R4 = -CH2OH
h
R4 = -COOH
Scheme 1. Reagents and conditions: (a) Ac2O/Et3N; (b) FeSO4/NH4OH; (c) NaNO2/fluoroboric acid; ferrocene/acetone; (d) EDC, DMAP, HOBt/
DMF; (e) BMS/THF; (f) H2, Pd/C/MeOH; (g) LiAlH4/THF; (h) NaOH/MeOH (1:1).
Structures of target compounds 10–42, including R1, R2, R3 and N-alkyl, are found in Tables 1–3.
d
For general structure 8 with R4 ¼ COOMe : 7a ! 10
For general structure 8 with R4 ¼ COOH : 7a=b d!þh 11; 16; 25; 29
For general structure 10 with R4 ¼ COOMe : 7a=b d!þe 14; 17; 26; 30
For general structure 10 with R4 ¼ COOH : 7a=b=c dþ!eþh 12; 15; 18; 20; 27; 31; 33; 35; 37
For general structure 10 with R4 ¼ CH2OH : 7a=b=c dþeþhþg
!
13; 19; 21; 28; 32; 34; 36; 38
f
For R3 ¼ OH; R4 ¼ COOH : 16; 18; 20 ! 22; 23; 24
g
For seco PBT analogs: 5c d!þh 39 ! 40 ! 41
e
compounds into drugs. In addition, by opening the
indolizidine ring, polar chemical moieties can easily be
introduced into the phenanthrene skeleton in
conjunction with Stærk’s proposal.
acetic acid (4a,b) in the presence of Ac2O (Perkin con-
densation) to yield an intermediate nitro-substituted cin-
namic acid (5a–c). The nitro group of 5 was converted to
an amine using ammoniacal ferrous sulfate (FeSO4) to
provide the amino-substituted cinnamic acid (6a–c).
Phenanthrenes 7a–c, which are the key intermediates
in our method, were formed by an improved free-radical
Pschorr cyclization26 in high yield. Compound 6 was
treated with sodium nitrite in 48% fluoroboric acid,
and the resulting diazonium tetrafluoroborate was then
efficiently cyclized to the phenanthrene-9-carboxylic
acid (7) using catalytic ferrocene. Intermediates 7a–c
were then condensed with the appropriate protected cyc-
lic or acyclic amino acid in the presence of N-(3-dimeth-
Therefore, as part of our studies in plant-derived antitu-
mor agents, we have initiated the design and synthesis of
new tylophorine analogs. This paper describes our initial
design and synthesis of novel polar PBTs, cytotoxicity
evaluation against the human A549 lung cancer cell line
(Table 1), and structure–activity relationships (SAR) of
this new compound class. The goal of this study is to
generate and optimize phenanthrene derivatives as
promising clinical trial candidates for treating cancer.
ylaminopropyl)-N0-ethylcarbodiimide
hydrochloride
(EDC), 4-(dimethylamino)pyridine (DMAP), and
1-hydroxybenzotriazole (HOBT) to give the amido-
substituted phenanthrene (8). These mild conditions
avoid the production of degradation products resulting
from the acidic instability of the methylenedioxy group.
Selective reduction of the amide carbonyl group to a
methylene was achieved by using borane-methyl sulfide
2. Chemistry
Compounds 10–42 were synthesized following the effi-
cient reported procedure26 outlined in Scheme 1. A com-
mercially available substitutedO-nitrobenzaldehyde
(3a,b) was treated with 4-methoxy or -benzyloxyphenyl

L. Wei et al. / Bioorg. Med. Chem. 14 (2006) 6560–6569
Table 3. SAR of seco-PBT analogs
6563
complex (BMS, 2.0 M solution in THF) to provide 9.
a
The C-9 side-chain methyl ester was converted to
carboxylic acid and hydroxymethyl groups by basic
hydrolysis and lithium aluminum hydride reduction,
respectively. The 6-phenolic analogs were prepared by
hydrogenolysis (H2, Pd/C) of the benzyloxy-protecting
group.
Compound
Structure
IC50 (lM)
N
HOOC
O
39
80
MeO
MeO
NO2
3. Results and discussion
OMe
Tables 1–3 summarize the structures of newly synthe-
sized PBTs (7a–c, 10–42) and their cytotoxic activity
(IC50) against the A549 lung cancer cell line. Etoposide
(VP-16) was used as the reference compound.
N
HOOC
40
41
42
45.2
11.7
52.2
All three intermediate phenanthrene-9-carboxylic acids
7a–c were inactive. Thus, the rigid, planar phenanthrene
system is not sufficient for cytotoxic activity, and an
appropriate C-9 side chain is crucial. However, the
phenanthrene system is required for cytotoxic activity,
as the seco analogs 39–42, which contain an active pro-
line side chain (see discussion below) but only a stilbene
skeleton, were also inactive.
MeO
MeO
NO2
OMe
HO
N
Active analogs contained both cyclic (pyrrole/proline,
piperidine/pipecolinic acid) and acylic (aminopentanoic,
aminohexanoic acids) nitrogen-containing side chains at
the C-9 position of phenanthrene. However, the linkage
between the nitrogen and the phenanthrene was very
critical. Reduction of the amide carbonyl to methylene
could dramatically increase the cytotoxic activity as
shown in the comparison of 16 (L-proline amide, car-
bonyl linkage, IC50 42.6 lM) and 18 (methylene linkage,
IC50 4.4 lM). This observation (carbonyl, unfavorable
or less favorable; methylene, favorable) also could be
seen in comparison of 11/12 and 25/27. Differences in
MeO
MeO
NO2
OMe
HO
N
MeO
MeO
OMe
a Etoposide (VP-16) used as positive control, IC50 = 1.4 lM.
Table 2. SAR of 2,3,6-trimethoxy-9-substituted PBT analogs
conformational constraint could possibly explain this
observation. The carbonyl is conjugated with the phen-
anthrene ring and, thus, extends the coplanarity, while
the methylene substituent can rotate freely around the
C–C bond. The former geometric restriction appears
to disfavor cytotoxic activity and may prevent the mol-
ecule from attaining the optimal conformation for bind-
ing to an assumed biological target. In addition, the
basicity of the nitrogen would differ between the amide
(carbonyl linkage) and amine (methylene linkage)
analogs.
OCH3
2
H3CO
3
R
9
H3CO
6
a,b
IC50 (lM)
Compound
R
7c
35
–COOH
–CH2NH(CH2)5COOH
–CH2NH(CH2)5CH2OH
NA
9.7
2.7
36
On the phenanthrene skeleton, changing the benzyloxy
moiety at the C-6 position to a hydroxy group was
extremely detrimental to activity (cf. 18/23 and 20/
24). In addition, active compounds with a 6-benzyloxy
moiety (15 and 18–21, IC50 1.3–4.4 lM) were generally
slightly less potent than corresponding analogs with a
6-methoxy group (27 and 31–34, IC50 0.16–2.1 lM).
Thus, the rank order of potency at the phenanthrene
C-6 position was methoxy > benzyloxy ꢀ hydroxyl,
suggesting that this position cannot tolerate the intro-
HOOC
N
37
38
9.7
6.3
HO
N
a Etoposide (VP-16) used as positive control, IC50 = 1.4 lM.
b NA, not active.

6564
L. Wei et al. / Bioorg. Med. Chem. 14 (2006) 6560–6569
duction of a polar moiety. A hydrophilic moiety may
be disfavored because a hydrophobic interaction oc-
curs at this position between the compound and bio-
logical target that is essential for enhanced cytotoxic
activity.
4. Conclusion
In this study, a total of 33 novel phenanthrene-based tyl-
ophorine derivatives (PBTs) were synthesized and
evaluated for cytotoxic activity against human A549
lung cancer cells. Among these compounds, N-(2,3-
methylenedioxy-6-methoxy-phenanthr-9-ylmethyl)-L-2-
piperidinemethanol (34) and N-(2,3-methylenedioxy-6-
methoxy-phenanthr-9-ylmethyl)-5-aminopentanol (28)
were the most potent compounds designed and synthe-
sized to date with IC50 values of 0.16 and 0.27 lM,
respectively. These two compounds incorporated all of
the favorable modifications identified to date. They pos-
sess a novel structure and showed remarkable IC50 val-
ues in the sub-micromolar range, comparable with
front-line antineoplastic drugs such as etoposide, sug-
gesting that this new compound class is worthy of fur-
ther development as potential antitumor clinical trial
candidates. We are extending these preliminary structur-
al and biological studies to include both additional com-
pounds and cancer cell lines.
Interestingly, active compounds with a 2,3-methylenedi-
oxy group (33 and 34, IC50 0.5 and 0.16 lM) were up to
40 times more potent than the corresponding com-
pounds with a 2,3-dimethoxy moiety (37 and 38, IC50
9.7 and 6.3 lM). Thus, the five-membered methylenedi-
oxy ring extension at the phenanthrene C2-C3 is quite
favorable for cytotoxic activity.
In the amino side chain, when the terminal carboxylic
acid group was masked as the methyl ester, the cytotoxic
activity was diminished drastically or abolished (e.g., 14/
15, 17/18, 26/27, and 30/31). However, reduction of the
carboxylic acid to the hydroxymethyl generally in-
creased activity, both in cyclic (18/19, 20/21, 31/32, 33/
34, and 37/38) and acyclic (12/13, 27/28, and 35/36) ana-
logs. Thus, the rank order of potency of the terminal po-
lar substituent was hydroxy > carboxylic acid ꢀ methyl
ester. Hydrogen bond donor/acceptor capability or
polarity may affect the activity.
5. Experimental
5.1. General information
In summary, the favorable modifications for these
novel PBTs are as follows. (1) A planar phenanthrene
system is required, but not sufficient for cytotoxic
activity. (2) A N-hydrophilic substituent at the C-9
position is essential for the enhanced cytotoxicity
and should be linked through a methylene rather than
a carbonyl group. (3) This C-9 N-hydrophilic substitu-
ent is ideal for the introduction of a polar moiety.
Analogs containing terminal carboxylic acid or
hydroxymethyl groups are more favorable than those
with methyl esters. (4) On the phenanthrene skeleton,
a methoxy substituent best fits both the steric and
electronic requirements at the C-6 position and is
preferred over benzyloxy and hydroxy groups. (5)
Adding a methylenedioxy ring at the 2,3 position of
the planar phenanthrene system can dramatically
enhance the cytotoxic activity and led to the most po-
tent derivatives.
Melting points were measured using a Fisher Johns melt-
ing apparatus without correction. Proton nuclear mag-
netic resonance (1H NMR) spectra were measured on a
400 MHz Varian Gemini 2000 spectrometer using TMS
as internal standard. The solvent used was CDCl3 unless
indicated. Mass spectra were recorded on a PE-Sciex
API-3000 LC/MS/MS instrument equipped with a Turbo
IonsSpray ion source. Elemental analyses were per-
formed by Atlantic Microlab, Inc., Norcross, GA. All ac-
tive target compounds were analyzed for C, H, N and
gave values within 0.4% of the theoretical values (Table
4). Thin-layer chromatography (TLC) was performed on
PLC silica gel 60 F254 plates (0.5 mm, Merck). Biotage
Flash+ and Isco Companion systems were used as medi-
um-pressure column chromatography. Silica gel (200–
400 mesh) from Aldrich, Inc. was used for column chro-
matography. 4-Benzyloxyphenylacetic acid and 3,4-
Table 4. Elemental analysis data for active PBT analogs
Compound
Chemical formula
Analysis
Anal. Calcd (%)
H
Found (%)
H
C
N
C
N
12
13
15
18
19
20
21
27
28
31
32
33
34
C
C
C
29H29O5N
29H31O4N
28H27O5N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
C, H, N
73.87
76.12
73.51
71.02
76.17
74.18
76.46
69.28
71.9
6.20
6.83
5.95
5.75
6.16
5.80
6.42
6.08
6.86
5.71
6.34
5.80
6.94
2.97
3.06
3.06
2.96
3.17
2.98
3.07
3.67
3.81
3.61
3.83
2.98
3.45
73.89
76.15
73.52
70.91
76.54
74.20
76.82
69.38
69.97
68.01
72.56
73.99
68.01
6.16
6.78
5.91
5.66
6.21
5.76
5.96
6.17
6.62
5.65
6.22
5.79
6.92
2.97
3.06
3.06
2.99
3.20
2.96
3.09
3.61
3.75
3.52
3.72
2.87
3.44
C28H25O5NÆ1.0 H20
28H27O4N
C29H27O5N
29H29O4N
C22H23O5N
22H25O4N
C22H21O5NÆ0.5 H20
22H23O4N
C23H23O5N
23H25O4NÆ1.5 H2O
C
C
C
68.03
72.31
74.18
67.96
C
C

L. Wei et al. / Bioorg. Med. Chem. 14 (2006) 6560–6569
6565
methylenedioxy-6-nitrobenzaldehyde were purchased
from TCI. L-Pipecolinic acid and isonipecotic acid were
commercially available from Lancaster. All other chemi-
cals were obtained from Aldrich, Inc. and Fisher, Inc.
mixture was stirred overnight at rt and partitioned be-
tween EtOAc and water. The organic layer was washed
with brine, saturated NaHCO3, and 1 N HCl, dried over
Na2SO4, filtered, and concentrated in vacuo. The crude
product was chromatographed using Biotage Flash and
Isco Companion systems using a 40 g silica cartridge
and EtOAc/hexane as eluant.
5.2. General synthetic procedures
5.2.1. Coupling reaction (a). A solution of 3,4-methylen-
edioxy-6-nitrobenzaldehyde (12 mmol), triethyl amine
(12 mmol), and 4-benzyloxy- or 4-methoxy-phenylacetic
acid (17 mmol) in anhydrous acetic anhydride (17 mL)
was refluxed with stirring under Ar for 40 min. Water
(30 mL) was added to the reaction mixture and the tem-
perature was maintained at 90–100 °C during the addi-
tion. The reaction mixture was cooled to rt and the solid
was collected by filtration and recrystallized from EtOH.
5.2.5. Carbonyl reduction reaction (e). To a stirred solu-
tion of 9-amido-substituted phenanthrene (2 mmol) in
THF (20 mL) was added dropwise borane-methyl sul-
fide (BMS) (4 mL, 2.0 M solution in THF). The reaction
mixture was stirred at rt overnight and quenched with
1 N HCl. THF was removed in vacuo, and the residue
was partitioned between CH2Cl2 and water. The organic
layer was dried, filtered, and evaporated to afford the
target product. The crude product was chromato-
graphed using Biotage Flash and Isco Companion sys-
tems using MeOH/CH2Cl2 as eluant.
5.2.2. Reduction of the nitro group (b). To a solution of
the nitrocinnamic acid (7 mmol) in 10% aqueous
NH4OH (100 mL) was added ferrous sulfate heptahy-
drate (15 g) dissolved in distilled water (100 mL) and
concentrated aqueous NH4OH (100 mL). The reaction
mixture was refluxed for 1.5 h, cooled to 40 °C, filtered
through Celite, and acidified with HOAc (100 mL).
The resulting solid was collected by filtration and recrys-
tallized from EtOH to yield the aminocinnamic acid.
5.2.6. Catalytic hydrogenolysis of benzyl group (g). A
solution of benzyloxy derivative (1 mmol) and Pd/C
(10%) was hydrogenated in a Parr apparatus (30 psi)
for 2 h. The reaction mixture was filtered through Celite,
and filtrate was concentrated in vacuo and was chro-
matographed using Biotage Flash and Isco Companion
systems using MeOH/CH2Cl2 as eluant.
5.2.3. Ring closure (c). A solution composed of the
aminocinnamic acid (3 mmol), NaOH (33 mmol), and
NaNO2 in water (10 mL) was added dropwise over
30 min with stirring to 48% fluoroboric acid (43 mmol)
at 0–5 °C. The mixture was stirred for 1 h, after which
sat. aqueous sulfamic acid was added until the mixture
tested negative to starch-iodide paper. The crude solid
was collected by filtration, dissolved in anhydrous ace-
tone (10 mL), and then added dropwise to ferrocene
(0.06 g, 0.3 mmol) in anhydrous acetone with stirring
over a 15 min period at rt. After an additional 15 min
of stirring, the green reaction mixture was added to
water (100 mL). A light-yellow precipitate was collected
and the trace amount of ferrocene was removed in vacuo
to afford the phenanthrene-9-carboxylic acid.
5.2.7. LiAlH4 reduction (g). To a suspension of methyl
ester (1 mmol) in anhydrous THF (15 mL) was added
LiAlH4 (1 g) in portions at 0 °C. The reaction mixture
was warmed to rt and heated to reflux for 4 h. After
cooling to 0 °C, the reaction mixture was quenched with
MeOH. 10% Rochelle salt was added to the mixture,
which was then extracted with water and 10% MeOH/
CH2Cl2. The organic layer was dried over Na2SO4,
and the crude product was chromatographed using Bio-
tage Flash and Isco Companion systems using MeOH/
CH2Cl2 as eluant.
5.2.8. Methyl ester hydrolysis reaction (h). A solution of
ester in a 1:1 mixture of 4 N NaOH and MeOH was re-
fluxed for 4 h. The reaction mixture was acidified and
partitioned between 10% MeOH/CH2Cl2 and 1 N HCl.
The organic layer was dried over Na2SO4, filtered, and
evaporated. The crude product was chromatographed
using Biotage Flash and Isco Companion systems using
MeOH/CH2Cl2 as eluant.
5.2.4. Peptide bond condensation reaction (d). To a solu-
tion of phenanthrene-9-carboxylic acid (4 mmol),
4-(dimethylamino) pyridine (DMAP) (2 mmol), and
1-hydroxybenzotriazole (HOBT) (4 mmol) in DMF
(20 mL) was added 4-methylmorpholine (NMM)
(1.03 mL). After the mixture was stirred at 0 °C for
15 min, N-(3-dimethylaminopropyl)-N0-ethylcarbodii-
mide hydrochloride (EDC) (4.4 mmol) was added in
portions. After 30 min stirring, the appropriate amino
acid methyl esterꢀ (4.4 mmol) was added. The reaction
5.2.9. 2,3-Methylenedioxy-6-benzyloxy-phenanthrene-9-
carboxylic acid (7a). 75% yield over two steps; mp
263–265 °C; 1H NMR (400.13 MHz)
d 8.40 (d,
J = 4 Hz, 1H), 7.92 (d, J = 2 Hz, 1H), 7.88 (s, 1H),
7.52 (m, 2H), 7.38 (m, 5H), 7.26 (dd, J = 4 Hz, 2 Hz,
1H), 6.14 (d, J = 4 Hz, 2H), 5.35 (d, J = 7 Hz, 2H);
ESI MS m/z: 373 (M+H)+.
ꢀ
For a cycloalkylamino acid, SOCl2 (0.4 mL) was added dropwise to
the amino acid (4 mmol) in anhydrous MeOH (4 mL) at À30 °C. The
reaction mixture was warmed to rt and refluxed for 1 h. For an
acyclic alkylamino acid, acetyl chloride (0.45 mL) was added
dropwise to anhydrous MeOH (3 mL) at 0 °C. After 10 min stirring,
the amino acid was added to the solution in portions. The mixture
was warmed to rt and refluxed for 2 h. Then the solvent was removed
in vacuo and the resulting methyl ester was used without further
purification.
5.2.10. 2,3-Methylenedioxy-6-methoxy-phenanthrene-9-
carboxylic acid (7b). 78% yield; white powder; mp 293–
295 °C; 1H NMR (400.13 MHz) d 7.67 (s, 1H), 7.60
(d, J = 4 Hz, 1H), 7.22 (dd, J = 4 Hz, 2 Hz, 1H), 6.92
(s, 1H), 6.89 (d, J = 2 Hz, 1H), 6.70 (s, 1H), 5.98 (s,
2H), 3.79 (s, 3H); ESI MS m/z: 297 (M+H)+.

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L. Wei et al. / Bioorg. Med. Chem. 14 (2006) 6560–6569
5.2.11. 2,3,6-Trimethoxyphenanthrene-9-carboxylic acid
(7c). 79% yield; white powder; mp 241–243 °C; 1H NMR
(400.13 MHz) d 8.93 (d, J = 4 Hz, 1H), 8.33 (s, 1H), 7.84
(d, J = 2Hz, 1H), 7.82 (s, 1H), 7.23 (s, 1H), 7.22 (dd,
J = 4 Hz, 2 Hz, 1H), 4.07 (s, 3H), 3.98 (s,3H), 3.97 (s,
3H); ESI MS m/z: 314 (M+H)+.
5.2.17. N-(2,3-Methylenedioxy-6-benzyloxy-phenanthr-9-
ylmethyl)-5-aminopentanoic acid (15). General procedure
h from 14 (93%); white powder, recrystallized from
EtOH to give white needles; mp 197–199 °C; H NMR
(400.13 MHz) d 7.89 (d, J = 4 Hz, 1H), 7.88 (d,
J = 2 Hz, 1H), 7.77 (s, 1H), 7.46 (s, 1H), 7.45 (d,
J = 4 Hz, 2H), 7.34 (t, J = 4 Hz, 2H), 7.29 (dd,
J = 4 Hz, 2 Hz, 1H), 7.27 (m, 1H), 7.16 (s, 1H), 6.05
(s, 2H), 5.21 (s, 2H), 4.42 (s, 2H), 2.89 (m, 2H), 2.27
(t, J = 6 Hz, 2H), 1.89 (m, 2H), 1.68 (m, 2H); ESI MS
m/z: 458 (M+H)+. Anal. (C28H27O5N) C, H, N.
1
5.2.12. Methyl N-(2,3-methylenedioxy-6-benzyloxy-phen-
anthr-9-ylcarbonyl)-6-aminohexanoate (10). General pro-
cedure d from 7a and methyl 6-aminohexanoate (92%);
colorless syrup; 1H NMR (400.13 MHz) d 8.09 (d,
J = 4 Hz, 1H), 7.81 (d, J = 2 Hz, 1H), 7.82 (s, 1H),
7.74 (d, J = 4 Hz, 2H), 7.45 (s, 1H), 7.33 (t, J = 4 Hz,
2H), 7.26 (m, 1H), 7.20 (dd, J = 4 Hz, 2 Hz, 1H), 7.09
(s, 1H), 6.03 (s, 2H), 5.18 (s, 2H), 3.41(s, 3H), 3.28 (m,
2H), 2.28 (t, J = 6 Hz, 2H), 1.61 (m, 2H), 1.56 (m,
2H), 1.38 (m, 2H); ESI MS m/z: 500 (M+H)+.
5.2.18. N-(2,3-Methylenedioxy-6-benzyloxy-phenanthr-9-
ylcarbonyl)-L-proline (16). General procedures d and h
from 7a and L-proline methyl ester (95%); white powder;
mp 221–223 °C; 1H NMR (400.13 MHz) d 8.00 (d,
J = 4Hz, 1H), 7.85 (d, J = 2 Hz, 1H), 7.81 (s, 1H), 7.46
(s, 1H), 7.45 (d, J = 4 Hz, 2H), 7.36 (t, J = 4 Hz, 2H),
7.30 (m, 1H), 7.23 (dd, J = 4 Hz, 2 Hz, 1H), 7.13 (s,
1H), 6.04 (s, 2H), 5.19 (s, 2H), 4.71 (t, J = 7 Hz, 1H),
3.62 (m, 2H), 2.21 (m, 2H), 1.83 (m, 2H); ESI MS m/z:
470 (M+H)+.
5.2.13. N-(2,3-Methylenedioxy-6-benzyloxy-phenanthr-9-
ylcarbonyl)-6-aminohexanoic acid (11). General proce-
dure h from 10 (100%); white needles; mp 198–200 °C;
1H NMR (400.13 MHz) d 8.06 (d, J = 4 Hz, 1H), 7.79
(d, J = 2 Hz, 1H), 7.75 (s, 1H), 7.48 (s, 1H), 7.40 (d,
J = 4 Hz, 2H), 7.30 (t, J = 4 Hz, 2H), 7.23 (m, 1H),
7.16 (dd, J = 4 Hz, 2 Hz, 1H), 7.09 (s, 1H), 6.00 (s,
2H), 5.15 (s, 2H), 3.23 (m, 2H), 2.23 (t, J = 6 Hz, 2H),
1.58 (m, 4H), 1.36 (m, 2H); ESI MS m/z: 486 (M+H)+.
5.2.19. N-(2,3-Methylenedioxy-6-benzyloxy-phenanthr-9-
ylmethyl)-L-proline methyl ester (17). General procedures
d and e from 7a and L-proline methyl ester (85%); brown
syrup; 1H NMR (400.13 MHz) d 7.97 (d, J = 4 Hz, 1H),
7.83 (d, J = 2 Hz, 1H), 7.80 (s, 1H), 7.71 (s, 1H), 7.59 (d,
J = 4 Hz, 2H), 7.47 (t, J = 4 Hz, 2H), 7.36 (m, 1H), 7.22
(dd, J = 4 Hz, 2 Hz, 1H), 7.11 (s, 1H), 6.06 (s, 2H), 5.20
(s, 2H), 4.75 (s, 2H), 3.67 (s, 3H), 3.26 (d, J = 17 Hz,
1H), 2.45 (m, 2H), 1.96 (m, 2H), 1.68 (m, 2H); ESI
MS m/z: 470 (M+H)+.
5.2.14. N-(2,3-Methylenedioxy-6-benzyloxy-phenanthr-9-
ylmethyl)-6-aminohexanoic acid (12). General procedures
e and h from 10 (90%); white powder, recrystallized from
1
EtOH to give white needles; mp 184–186 °C; H NMR
(400.13 MHz) d 7.90 (d, J = 4 Hz, 1H), 7.87 (d,
J = 2 Hz, 1H), 7.77 (s, 1H), 7.60 (s, 1H), 7.44 (d,
J = 4 Hz, 2H), 7.34 (t, J = 4 Hz, 2H), 7.30 (dd,
J = 4 Hz, 2 Hz, 1H), 7.28 (m, 1H), 7.16 (s, 1H), 6.03 (s,
2H), 5.19 (s, 2H), 4.46 (s, 2H), 2.83 (m, 2H), 2.26 (t,
J = 6 Hz, 2H), 1.90 (m,2H), 1.68 (m, 2H), 1.28 (m, 2H);
ESI MS m/z: 472 (M+H)+. Anal. (C29H29O5N) C, H, N.
5.2.20. N-(2,3-Methylenedioxy-6-benzyloxy-phenanthr-9-
ylmethyl)-L-proline (18). General procedure h from 17
(100%); white powder; mp 147–149 °C; 1H NMR
(400.13 MHz) d 8.11 (d, J = 4 Hz, 1H), 7.84 (d,
J = 2 Hz, 1H), 7.75 (s, 1H), 7.42 (s, 1H), 7.40 (d,
J = 4 Hz, 2H), 7.30 (t, J = 4 Hz, 2H), 7.27 (m, 1H),
7.23 (dd, J = 4 Hz, 2 Hz, 1H), 7.14 (s, 1H), 6.01 (s,
2H), 5.16 (s, 2H), 4.30 (s, 2H), 3.24 (m, 1H), 2.48 (m,
2H), 2.0 (m, 2H), 1.68 (m, 2H); ESI MS m/z: 456
(M+H)+. Anal. (C28H25O5NÆ1.0 H2O) C, H, N.
5.2.15. N-(2,3-Methylenedioxy-6-benzyloxy-phenanthr-9-
ylmethyl)-6-aminohexanol (13). General procedure g
from 12 (95%); white powder; mp 175–177 °C; 1H
NMR (400.13 MHz) d 7.90 (d, J = 4 Hz, 1H), 7.86 (d,
J = 2 Hz, 1H), 7.76 (s, 1H), 7.45 (s, 1H), 7.44 (d,
J = 4 Hz, 2H), 7.32 (t, J = 4 Hz, 2H), 7.26 (m, 1H), 7.24
(dd, J = 4 Hz, 2 Hz, 1H), 7.14 (s, 1H), 6.03 (s, 2H), 5.22
(s, 2H), 4.24 (s, 2H), 3.53 (t, J = 6 Hz, 2H), 2.72 (t,
J = 6 Hz, 2H), 1.58 (m, 2H), 1.47 (m, 2H), 1.29 (m, 4H);
ESI MS m/z: 458 (M+H)+. Anal. (C29H31O4N) C, H, N.
5.2.21. N-(2,3-Methylenedioxy-6-benzyloxy-phenanthr-9-
ylmethyl)-L-prolinol (19). General procedure g from 18
(95%); colorless oil, recrystallization from EtOH gave
1
white powder; mp 122–124 °C; H NMR (400.13 MHz)
d 8.13 (d, J = 4 Hz, 1H), 7.90 (d, J = 2 Hz, 1H), 7.84
(s, 1H), 7.51 (d, J = 4 Hz, 2H), 7.42 (s, 1H), 7.41 (t,
J = 4 Hz, 2H), 7.34 (m, 1H), 7.29 (dd, J = 4 Hz, 2 Hz,
1H), 7.14 (s, 1H), 6.06 (s, 2H), 5.24 (s, 2H), 4.36 (d,
J = 7 Hz, 2H), 3.78 (d, J = 17 Hz, 2H), 3.41 (m, 1H),
2.42 (m, 2H), 1.83 (m, 2H), 1.66 (m, 6H); ESI MS m/z:
442 (M+H)+. Anal. (C28H27O4N) C, H, N.
5.2.16. Methyl N-(2,3-methylenedioxy-6-benzyloxy-phen-
anthr-9-ylmethyl)-5-aminopentanoate
(14).
General
procedures d and e from 7a and methyl 5-aminopent-
anoate (93%); colorless syrup; 1H NMR (400.13
MHz) d 7.97 (d, J = 4 Hz, 1H), 7.83 (d, J = 2 Hz, 1H),
7.77 (s, 1H), 7.46 (s, 1H), 7.45 (d, J = 4 Hz, 2H), 7.35
(t, J = 4 Hz, 2H), 7.27 (m, 1H), 7.20 (dd, J = 4 Hz,
2 Hz, 1H), 7.07 (s, 1H), 6.01 (s, 2H), 5.17 (s, 2H), 4.95
(s, 2H), 3.50 (s, 3H), 3.01 (m, 2H), 2.42 (t, J = 6 Hz,
2H), 1.67 (m, 2H), 1.59 (m, 2H); ESI MS m/z: 472
(M+H)+.
5.2.22. N-(2,3-Methylenedioxy-6-benzyloxy-phenanthr-9-
ylmethyl)-L-2-piperidinecarboxylic acid (20). General
procedures d, e, and h from 7a and methyl L-2-piperidin-
1
ecarboxylate (83%); white powder; mp 168–170 °C; H
NMR (400.13 MHz) d 8.26 (d, J = 4 Hz, 1H), 7.85 (d,
J = 2 Hz, 1H), 7.77 (s, 1H), 7.56 (s, 1H), 7.45 (d,

L. Wei et al. / Bioorg. Med. Chem. 14 (2006) 6560–6569
6567
J = 4 Hz, 2H), 7.35 (t, J = 4 Hz, 2H), 7.30 (m, 1H), 7.26
(dd, J = 4 Hz, 2 Hz, 1H), 7.12 (s, 1H), 6.05 (s, 2H), 5.20
(s, 2H), 4.24 (s, 2H), 3.16 (m, 1H), 2.26 (m, 2H), 1.80 (m,
2H), 1.64 (m, 4H); ESI MS m/z: 470 (M+H)+. Anal.
(C29H27O5N) C, H, N.
5.2.29. N-(2,3-Methylenedioxy-6-methoxy-phenanthr-9-
ylmethyl)-5-aminopentanoic acid (27). General procedure
h from 26 (99%); white powder; mp 145–146 °C; 1H
NMR (400.13 MHz) d 8.06 (d, J = 4 Hz, 1H), 7.79 (s,
1H), 7.75 (d, J = 2 Hz, 1H), 7.48 (s, 1H), 7.22 (dd,
J = 4 Hz, 2 Hz, 1H), 7.10 (s, 1H), 6.03 (s, 2H), 4.02 (s,
2H), 3.82 (s, 3H), 2.75 (m, 2H), 2.23 (t, J = 6 Hz, 2H),
1.53 (m, 2H), 1.44 (m, 2H); ESI MS m/z: 382 (M+H)+.
Anal. (C22H23O5N) C, H, N.
5.2.23. N-(2,3-Methylenedioxy-6-benzyloxy-phenanthr-9-
ylmethyl)-L-piperidinemethanol (21). General procedure
g from 20 (95%); light brown oil, recrystallization from
1
EtOH gave white powder; mp 151–152 °C ; H NMR
(400.13 MHz) d 8.17 (d, J = 4 Hz, 1H), 7.89 (d,
J = 2 Hz, 1H), 7.83 (s, 1H), 7.51 (d, J = 4 Hz, 2H),
7.43 (s, 1H), 7.40 (t, J = 4 Hz, 2H), 7.34 (m, 1H), 7.27
(dd, J = 4 Hz, 2 Hz, 1H), 7.14 (s, 1H), 6.06 (s, 2H),
5.23 (s, 2H), 4.45 (d, J = 7 Hz, 2H), 3.46 (d, J = 17 Hz,
2H), 2.85 (m, 1H), 2.37 (m, 2H), 1.69 (m, 6H); ESI
MS m/z: 456 (M+H)+. Anal. (C29H29O4N) C, H, N.
5.2.30. N-(2,3-Methylenedioxy-6-methoxy-phenanthr-9-
ylmethyl)-5-aminopentanol (28). General procedure g
from 27 (97%); colorless oil, recrystallization from
1
EtOH gave white powder; mp 125–126 °C; H NMR
(400.13 MHz) d 8.00 (d, J = 4 Hz, 1H), 7.76 (s, 1H),
7.72 (d, J = 2 Hz, 1H), 7.44 (s, 1H), 7.13 (dd, J = 4 Hz,
2 Hz, 1H), 7.05 (s, 1H), 6.04 (s, 2H), 3.95 (s, 2H), 3.93
(s, 3H), 3.58 (t, J = 6 Hz, 2H), 2.75 (m, 2H), 1.55 (m,
2H), 1.44 (m, 2H), 1.30 (m, 2H); ESI MS m/z: 368
(M+H)+. Anal. (C22H25O4N) C, H, N.
5.2.24. N-(2,3-Methylenedioxy-6-hydroxy-phenanthr-9-
ylcarbonyl)-L-proline (22). General procedure f from 16
(92%); white powder; mp 230–231 °C; 1H NMR
(400.13 MHz) d 7.73 (d, J = 4 Hz, 1H), 7.67 (s, 1H),
7.50 (d, J = 2 Hz, 1H), 7.35 (s, 1H), 7.21 (dd, J = 4 Hz,
2 Hz, 1H), 7.06 (m, 1H), 6.01 (s, 2H), 4.67 (t,
J = 7 Hz, 1H), 3.22 (m, 2H), 2.38 (m, 2H), 2.18 (m,
2H), 1.86 (m, 2H); ESI MS m/z: 380 (M+H)+.
5.2.31. N-(2,3-Methylenedioxy-6-methoxy-phenanthr-9-
ylcarbonyl)-L-proline (29). General procedures d and h
from 7b and L-proline methyl ester (93%); white powder;
mp 207–208 °C; 1H NMR (400.13 MHz) d 7.78(d,
J = 4 Hz, 1H), 7.70 (s, 1H), 7.54 (d, J = 2 Hz, 1H), 7.35
(s, 1H), 7.20 (dd, J = 4 Hz, 2 Hz, 1H), 7.14 (m, 1H),
6.01 (s, 2H), 4.67 (m, 1H), 3.90 (s, 3H), 3.22 (m, 2H),
2.38 (m, 2H), 1.86 (m, 2H); ESI MS m/z: 394 (M+H)+.
5.2.25. N-(2,3-Methylenedioxy-6-hydroxy-phenanthr-9-
ylmethyl)-L-proline (23). General procedure f from 18
(95%); white powder; mp 205–206 °C; 1H NMR
(400.13 MHz) d 8.07 (d, J = 4 Hz, 1H), 7.82 (s, 1H),
7.80 (d, J = 2 Hz, 1H), 7.42 (s, 1H), 7.21 (dd, J = 4 Hz,
2 Hz, 1H), 7.10 (s, 1H), 6.04 (s, 2H), 4.09 (s, 2H), 3.16
(m, 1H), 2.38 (m, 2H), 2.23 (m, 2H), 1.95 (m, 2H);
ESI MS m/z: 366 (M+H)+.
5.2.32. N-(2,3-Methylenedioxy-6-methoxy-phenanthr-9-
ylmethyl)-L-proline methyl ester (30). General procedures
d and e from 7a (85%); white syrup; 1H NMR
(400.13 MHz) d 8.20 (d, J = 4 Hz, 1H), 7.80 (s, 1H),
7.70 (d, J = 2 Hz, 1H), 7.52 (s, 1H), 7.21 (dd, J = 4 Hz,
2 Hz, 1H), 7.13 (s, 1H), 6.05 (s, 2H), 4.02 (s, 2H), 3.89
(s, 3H), 3.67 (s, 3H), 3.14 (m, 1H), 2.32 (m, 2H), 2.14
(m, 2H), 1.86 (m, 2H); ESI MS m/z: 394 (M+H)+.
5.2.26. N-(2,3-Methylenedioxy-6-hydroxy-phenanthr-9-
ylmethyl)-L-2-piperidinecarboxylic acid (24). General
procedure f from 20 (95%); white powder; mp 211–
213 °C; 1H NMR (400.13 MHz) d 8.07 (d, J = 4 Hz,
1H), 7.80 (s, 1H), 7.76 (d, J = 2 Hz, 1H), 7.50 (s, 1H),
7.22 (dd, J = 4 Hz, 2 Hz, 1H), 7.12 (s, 1H), 6.05 (s,
2H),4.06 (s, 2H), 3.12 (m, 1H), 2.38 (m, 2H), 1.88 (m,
2H), 1.64 (m, 4H); ESI MS m/z: 380 (M+H)+.
5.2.33. N-(2,3-Methylenedioxy-6-methoxy-phenanthr-9-
ylmethyl)-L-proline (31). General procedure h from 30
(100%); white powder; mp 145–146 °C; 1H NMR
(400.13 MHz) d 8.21 (d, J = 4 Hz, 1H), 7.77 (s, 1H),
7.72 (d, J = 2 Hz, 1H), 7.56 (s, 1H), 7.23 (dd, J = 4 Hz,
2 Hz, 1H), 7.10 (s, 1H), 6.04 (s, 2H), 4.05 (s, 2H), 3.96
(s, 3H), 3.16 (m, 1H), 2.36 (m, 2H), 2.14 (m, 2H), 1.81
(m, 2H); ESI MS m/z: 380 (M+H)+. Anal.
(C22H21O5NÆ0.5 H2O) C, H, N.
5.2.27. N-(2,3-Methylenedioxy-6-methoxy-phenanthr-9-
ylcarbonyl)-5-aminopentanoic acid (25). General proce-
dures d and h from 7b and methyl 5-aminopentanoate
(100%); white powder; mp 137–138 °C; 1H NMR
(400.13 MHz) d 7.92 (d, J = 4 Hz, 1H), 7.82 (s, 1H),
7.75 (s, 1H), 7.71 (d, J = 2 Hz, 1H), 7.13 (dd, J = 4 Hz,
2 Hz, 1H), 7.09 (s, 1H), 6.00 (s, 2H), 3.89 (s, 3H), 3.23
(m, 2H), 2.23 (t, J = 6 Hz, 2H), 1.58 (m, 4H); ESI MS
m/z: 412 (M+H)+.
5.2.34. N-(2,3-Methylenedioxy-6-methoxy-phenanthr-9-
ylmethyl)-L-prolinol (32). General procedure g from 31
(95%); white powder; mp 138–139 °C; 1H NMR
(400.13 MHz) d 8.16 (d, J = 4 Hz, 1H), 7.88 (s, 1H),
7.80 (d, J = 2 Hz, 1H), 7.46 (s, 1H), 7.23 (dd, J = 4 Hz,
2 Hz, 1H), 7.14 (s, 1H), 6.07 (s, 2H), 4.03 (s, 2H), 3.94
(s, 3H), 3.70 (d, J = 17 Hz, 2H), 3.16 (m, 1H), 2.38 (m,
2H), 2.23 (m, 2H), 1.95 (m, 2H); ESI MS m/z: 366
(M+H)+. Anal. (C22H23O4N) C, H, N.
5.2.28. Methyl N-(2,3-methylenedioxy-6-methoxy-phen-
anthr-9-ylmethyl)-5-aminopentanoate (26). General pro-
cedures d and e from 7b (90%); colorless syrup; 1H
NMR (400.13 MHz) d 8.10 (d, J = 4 Hz, 1H), 7.82 (s,
1H), 7.70 (d, J = 2 Hz, 1H), 7.52 (s, 1H), 7.12 (dd,
J = 4 Hz, 2 Hz, 1H), 7.08 (s, 1H), 6.04 (s, 2H), 3.89 (s,
3H), 3.80 (s, 2H), 3.56 (s, 3H), 2.80 (m, 2H), 2.33 (t,
J = 6 Hz, 2H), 1.64 (m, 4H); ESI MS m/z: 396 (M+H)+.
5.2.35. N-(2,3-Methylenedioxy-6-methoxy-phenanthr-9-
ylmethyl)-L-2-piperidinecarboxylic acid (33). General
procedures d, e, and h from 7b and methyl L-2-piperidin-
1
ecarboxylate (80%); white powder; mp 171–172 °C; H

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L. Wei et al. / Bioorg. Med. Chem. 14 (2006) 6560–6569
NMR (400.13 MHz) d 8.21 (d, J = 4Hz, 1H), 7.77 (s,
1H), 7.72 (d, J = 2 Hz, 1H), 7.56 (s, 1H), 7.23 (dd,
J = 4 Hz, 2 Hz, 1H), 7.10 (s, 1H), 6.04 (s, 2H), 4.08 (s,
2H), 3.96 (s, 3H), 3.08 (m, 1H), 2.38 (m, 2H), 1.84 (m,
2H), 1.60 (m, 4H); ESI MS m/z: 394 (M+H)+. Anal.
(C23H23O5N) C, H, N.
3H), 3.41(t, J = 7 Hz, 2H), 2.22 (m, 2H), 1.92 (m, 2H);
ESI MS m/z: 443 (M+H)+.
5.2.42.
1-[3-(4,5-Dimethoxy-2-nitrophenyl)-2-(4-meth-
oxyphenyl)-allyl]-pyrrolidine-2-carboxylic acid (40). Gen-
eral procedure e from 39 (90%); brown syrup; 1H NMR
(400.13 MHz) d 7.61 (s, 1H), 7.24 (d, J = 4Hz, 2H), 7.01
(s, 1H), 6.77 (d, J = 4 Hz, 2H), 6.64 (s, 1H), 4.36 (d,
J = 7 Hz, 2H), 3.89 (s, 3H), 3.72 (t, J = 7 Hz, 1H), 3.69
(s, 3H), 3.42 (s, 3H), 3.24 (m, 2H), 2.18 (m, 2H), 1.84
(m, 2H); ESI MS m/z: 429 (M+H)+.
5.2.36. N-(2,3-Methylenedioxy-6-methoxy-phenanthr-9-
ylmethyl)-L-2-piperidinemethanol (34). General proce-
dure g from 33 (98%); white powder; mp 155–157 °C;
1H NMR (400.13 MHz) d 8.16 (d, J = 4 Hz, 1H), 7.88
(s, 1H), 7.80 (d, J = 2 Hz, 1H), 7.47 (s, 1H), 7.22 (dd,
J = 4 Hz, 2 Hz, 1H), 7.15 (s, 1H), 6.07 (s, 2H), 4.06 (s,
2H), 3.98 (s, 3H), 3.68 (d, J = 7 Hz, 2H), 2.87 (m,
1H), 2.40 (m, 2H), 1.64 (m, 6H); ESI MS m/z: 380
(M+H)+. Anal. (C23H25O4NÆ1.5 H2O) C, H, N.
5.2.43. {1-[3-(4,5-Dimethoxy-2-nitrophenyl)-2-(4-meth-
oxyphenyl)-allyl]-pyrrolidin-2-yl}-methanol (41). General
procedure g from 40 (90%); dark oil; 1H NMR
(400.13 MHz) d 7.59 (s, 1H), 7.23 (d, J = 4 Hz, 2H),
7.00 (s, 1H), 6.71 (d, J = 4 Hz, 2H), 6.23 (s, 1H), 4.17
(d, J = 7 Hz, 2H), 3.89 (s, 3H), 3.78 (d, J = 7 Hz, 2H),
3.69 (s, 3H), 3.58 (m, 1H), 3.41 (s, 3H), 3.16 (m, 2H),
1.91 (m, 2H), 1.80 (m, 2H); ESI MS m/z: 415 (M+H)+.
5.2.37.
N-(2,3,6-Trimethoxyphenanthr-9-ylmethyl)-6-
aminohexanoic acid (35). General procedures d, e, and
h from 7b and methyl 6-aminohexanoate (77%); brown
powder; mp 133–134 °C; 1H NMR (400.13 MHz) d
7.90 (d, J = 4 Hz, 1H), 7.78 (s, 1H), 7.72 (d, J = 2 Hz,
1H), 7.52 (s, 1H), 7.20 (dd, J = 4 Hz, 2 Hz, 1H), 7.08
(s, 1H), 3.92 (s, 9H), 3.71 (s, 2H), 2.87 (m, 2H), 2.23
(m, 2H), 1.55 (m, 2H), 1.46 (m, 2H), 1.28 (m, 2H);
ESI MS m/z: 412 (M+H)+.
5.2.44.
{1-[3-(3,4-Dimethoxyphenyl)-2-(4-methoxyphe-
nyl)allyl]-pyrrolidin-2-yl}-methanol (42). Silmilar proce-
dure as 40 (77% for two steps); yellow syrup; 1H
NMR (400.13 MHz) d 7.11 (d, J = 5 Hz, 2H), 6.89 (d,
J = 5 Hz, 2H), 6.63 (s, 1H), 6.58 (d, J = 3 Hz, 2H),
6.39 (s, 1H), 4.06 (d, J = 7Hz, 2H), 3.90 (d, J = 6 Hz,
2H), 3.71 (s, 3H), 3.69 (s, 3H), 3.65 (m, 1H), 3.39 (s,
3H), 3.27 (m, 2H), 1.84 (m, 2H), 1.70 (m, 2H); ESI
MS m/z: 370 (M+H)+.
5.2.38. N-(2,3,6-Trimethoxyphenanthr-9-ylmethyl)-6-
aminohexanol (36). General procedure g from 35
1
(92%); yellow oil; H NMR (400.13 MHz) d 7.88 (d,
J = 4 Hz, 1H), 7.76 (s, 1H), 7.71 (d, J = 2 Hz, 1H),
7.44 (s, 1H), 7.13 (dd, J = 4 Hz, 2 Hz, 1H), 7.05 (s,
1H), 5.99 (s, 2H), 3.94 (s, 9H), 3.80 (s, 2H), 3.73 (t,
J = 6 Hz, 2H), 2.75 (m, 2H), 1.50 (m, 4H), 1.28 (m,
4H); ESI MS m/z: 398 (M+H)+.
5.3. Cell growth inhibition assay
The human A549 lung cancer cell line was used for the
cytotoxicity screening of PBT derivatives using the
cell-based sulforhodamine B (SRB) microtiter plate as-
say.28 Compound stock solutions were prepared in
DMSO with the final solvent concentration 62%
DMSO (v/v), a concentration without effect on cell rep-
lication. The cells were cultured at 37 °C in RPMI-1640
supplemented with 25 mM N-2-hydroxyethylpiperazine-
N0-2-ethanesulfonic acid (HEPES), 2% (w/v) sodium
bicarbonate, 10% (v/v) fetal bovine serum, and 100 lg/
mL kanamycin in a humidified atmosphere containing
5% CO2. Duration of compound exposure was 3 days.
The IC50 value (the concentration that reduced the cell
number by 50%) was interpolated from dose–response
data. Each test was performed in triplicate with varia-
tion less than 5%. The IC50 values determined in each
independent test varied less than 10%.
5.2.39. N-(2,3,6-Trimethoxyphenanthr-9-ylmethyl)-L-2-
piperidinecarboxylic acid (37). General procedures d, e,
and h from 7c and methyl L-2-piperidinecarboxylate
1
(75%); light yellow powder; mp 187–188 °C; H NMR
(400.13 MHz) d 8.21 (d, J = 4 Hz, 1H), 7.78 (s, 1H),
7.71 (d, J = 2 Hz, 1H), 7.56 (s, 1H), 7.23 (dd, J = 4 Hz,
2 Hz, 1H), 7.10 (s, 1H), 4.10 (s, 2H), 3.98 (s, 9H), 3.12
(m, 1H), 2.42 (m, 2H), 1.80 (m, 2H), 1.60 (m, 4H);
ESI MS m/z: 410 (M+H)+.
5.2.40. N-(2,3,6-Trimethoxyphenanthr-9-ylmethyl)-L-2-
piperidinemethanol (38). General procedure g from 37
(95%); brown oil, recrystallization from EtOH gave yel-
1
low powder; mp 135–137 °C; H NMR (400.13 MHz) d
8.21 (d, J = 4 Hz, 1H), 7.78 (s, 1H), 7.72 (d, J = 2 Hz,
1H), 7.50 (s, 1H), 7.20 (dd, J = 4 Hz, 2 Hz, 1H), 7.14
(s, 1H), 6.07 (s, 2H), 4.10 (s, 2H), 3.96 (s, 9H), 3.74 (d,
J = 17 Hz, 2H), 2.87 (m, 1H), 2.41 (m, 2H), 1.66 (m,
6H); ESI MS m/z: 396 (M+H)+.
Acknowledgment
This investigation was supported by Grant CA 17625
from National Cancer Institute awarded to K.H. Lee.
5.2.41.
1-[3-(4,5-Dimethoxy-2-nitrophenyl)-2-(4-meth-
oxyphenyl)-acryloyl]-pyrrolidine-2-carboxylic acid (39).
General procedures d and h from 5b and methyl pyrrol-
idine-2-carboxylate (90%); brown powder; mp 120–
122 °C; H NMR (400.13 MHz) d7.67 (s, 1H), 7.26 (d,
J = 4 Hz, 2H), 7.08 (s, 1H), 6.73 (d, J = 4Hz, 2H), 6.44
(s, 1H), 4.63 (t, J = 7 Hz, 1H), 3.92 (s, 6H), 3.73 (s,
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