Antitumor agents 288: Design, synthesis, SAR, and biological studies of novel heteroatom-incorporated antofine and cryptopleurine analogues as potent and selective antitumor agents

Article abstract of DOI:10.1021/jm200330s

Novel heteroatom-incorporated antofine and cryptopleurine analogues were designed, synthesized, and tested against a panel of five cancer cell lines. Two new S-13-oxo analogues (11 and 16) exhibited potent cell growth inhibition in vitro (GI₅₀: 9 nM and 20 nM). Interestingly, both compounds displayed improved selectivity among different cancer cell lines, in contrast to the natural products antofine and cryptopleurine. Mechanism of action (MOA) studies suggested that R-antofine promotes dysregulation of DNA replication during early S phase, while no similar effects were observed for 11 and 15 on corresponding replication initiation complexes. Compound 11 also showed greatly reduced cytotoxicity against normal cells and moderate antitumor activity against HT-29 human colorectal adenocarcinoma xenograft in mice without overt toxicity.

Full text of DOI:10.1021/jm200330s

ARTICLE
pubs.acs.org/jmc
Antitumor Agents 288: Design, Synthesis, SAR, and Biological Studies
of Novel Heteroatom-Incorporated Antofine and Cryptopleurine
Analogues as Potent and Selective Antitumor Agents
Xiaoming Yang,† Qian Shi,*,† Shuenn-Chen Yang,§ Chi-Yuan Chen,§ Sung-Liang Yu,||,3 Kenneth F. Bastow,‡
Susan L. Morris-Natschke,† Pei-Chi Wu,‡ Chin-Yu Lai,^ Tian-Shung Wu,O Shiow-Lin Pan,^
Che-Ming Teng,^ Jau-Chen Lin,§ Pan-Chyr Yang,*,^,§,# and Kuo-Hsiung Lee*,†,O
†Natural Products Research Laboratories and ‡Division of Medicinal Chemistry and Natural Products, Eshelman School of Pharmacy,
University of North Carolina, Chapel Hill, North Carolina 27599-7568, United States
§Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
Department of Clinical Laboratory Sciences and Medical Biotechnology, ^Department of Internal Medicine, College of Medicine, and
#Division of Genomic Medicine, Research Center for Medical Excellence, National Taiwan University, Taipei, Taiwan
3Department of Laboratory Medicine, National Taiwan University Hospital, Taipei, Taiwan
OChinese Medicine Research and Development Center, China Medical University and Hospital, Taichung 404, Taiwan
S Supporting Information
b
ABSTRACT: Novel heteroatom-incorporated antofine and cryptopleurine analogues were designed,
synthesized, and tested against a panel of five cancer cell lines. Two new S-13-oxo analogues (11 and
16) exhibited potent cell growth inhibition in vitro (GI50: 9 nM and 20 nM). Interestingly, both
compounds displayed improved selectivity among different cancer cell lines, in contrast to the natural
products antofine and cryptopleurine. Mechanism of action (MOA) studies suggested that R-antofine
promotes dysregulation of DNA replication during early S phase, while no similar effects were observed
for 11 and 15 on corresponding replication initiation complexes. Compound 11 also showed greatly
reduced cytotoxicity against normal cells and moderate antitumor activity against HT-29 human
colorectal adenocarcinoma xenograft in mice without overt toxicity.
’ INTRODUCTION
Phenanthroindolizidines and phenanthroquinolizidines,
which are natural alkaloids isolated from several plant species
such as the Asclepiadacae and Moracae family,1 have been
investigated intensively in recent years, due to their interesting
biological characteristics, including antimicrobial, antiangio-
genic, and anti-inflammatory effects,2ꢀ8 as well as significant
cytotoxicity as demonstrated in the NCI 60 cell-line assay
(Figure 1).9 Further studies have shown that these natural
Figure 1. Representative structures of phenanthroindolizidines and
products exhibit not only strong inhibitory activity against cancer
cell growth but also significant effects on cancer cells resistant or
cross-resistant to many anticancer drugs in the market.10 Thus,
this important class of chemical entities may potentially augment
our present arsenal of anticancer drugs.11,12
Although the biological target(s) and MOA of these natural
products are currently still unclear, some interesting findings
have been reported. A possible mechanism of action might be
inhibition of NF-kB signaling, a well-known pathway in the
antiapoptosis and survival of cancer cells, as well as regulation of
P-glycoprotein.13 Other hypotheses, such as inhibition of protein
synthesis during the chain elongation stage,14 targeting riboso-
mal subunits (low-affinity binding pockets have been identified
in the 40S, 60S, 70S, and 80S subunits),15ꢀ17 inhibition of
phenanthroquinolizidines.
hypoxia-inducible factor 1 (HIF-1),18 inhibition of thymidylate
synthase (TS) and dihydrofolate reductase (DHFR),19ꢀ21 sup-
pression of activator protein-1 (AP-1) and the cAMP response
element (CRE) signaling pathway, reduction of cell cycle reg-
ulatory proteins such as cyclin D1, cyclin B1, CDK1, CDK2, and
CDK4, etc.10 have also been reported. In addition, evidence
has suggested that certain structural analogues might not be
functional analogues,22 and thus, multiple biological targets
might exist.
Received: March 22, 2011
Published: June 13, 2011
r
2011 American Chemical Society
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Scheme 1a
a Reagents and conditions: (a) (Boc)2O, Et3N, CH2Cl2; (b) (i) Py SO3, DMSO, Et3N, CH2Cl2; (ii) RNH2, HOAc, NaBH3CN, MeOH; (iii) TFA,
3
CH2Cl2; (iv) K2CO3, MgSO4, HCHO, CH2Cl2.
Despite their promise, the potential development of antofine,
cryptopleurine, or related natural or synthetic alkaloids as
promising drug candidates has been limited, to a large degree,
by severe CNS toxicity, such as disorientation and ataxia,23 and
low natural availability. All of these factors combine to justify an
urgent need for improving the pharmacokinetic and pharmaco-
dynamic properties, such as polarity, of this compound class
through rational structural modifications. However, only limited
studies have been reported in this regard, e.g., introduction of a
hydroxy group at C14 of phenanthroindolizidines,24 construc-
tion of phenanthrene-based tylophorine analogues,25ꢀ27 and N
atom incorporation at C13a of tylophorine,28 probably due to the
lack of a convergent and efficient synthetic methodology. Re-
cently, our group reported a new strategy suitable for producing
numerous new phenanthroindolizidine and phenanthroquinoli-
zidine analogues with E-ring modifications.29 The versatility and
significance of this approach lie in the fact that it not only
facilitates the SAR study of E-ring variations but also, even more
importantly, generates a group of analogues carrying a hetero-
cyclic E-ring or other polar moieties to potentially overcome
CNS toxicity. CNS toxicity is closely associated with the blood
ꢀbrain barrier (BBB) penetration of a molecule, which is in large
part related to its physicochemical properties, such as lipophili-
city (cLogP) or polarity, molecular size and charge, polar surface
area (PSA), or hydrogen bonding potential, etc., although other
factors may also play a role.30ꢀ32 Herein, we report the design,
synthesis, in vitro anticancer activity, SAR, and mechanistic
studies of new antofine and cryptopleurine derivatives with a N
or O atom incorporated in the E-ring. The in vivo antitumor
activity for the most active compound (11) is also reported.
greater than 20 μM. Insertion of a single methylene group
between the N and phenyl ring (3a and 3j, R = CH2Ph) resulted
in greater activity (with the R isomer approximately 2-fold better
than the S isomer), while addition of a second methylene group
(3d, R = CH2CH2Ph) did not improve activity any further.
Generally, the compounds with an aliphatic amino moiety [cyclic
(3g), straight chain (3c, 3 h, and 3l), or branched chain (3b)]
showed slightly better activity than those bearing an aromatic
moiety. Compounds 3h and 3l (R = Me) showed the greatest
potency among all compounds, and the R isomer (3l) was
slightly more potent than its S enantiomer (3h). Conversely, 3i
(S isomer) was 2-fold more active than its R isomer (3k). These
data implied that introduction of a N atom at position C12 of
antofine might not improve the cytotoxicity against cancer cell
lines, even though it did increase the polarity as predicted by
PreADMET.33
Next, we studied the effect of N-incorporation in cryptopleur-
ine, both at positions C12 and C13. For the latter compound
series, 2 (S or R) was converted to 4 through oxidation with
Py SO3 and subsequent reductive amination using glycine methyl
3
ester hydrochloride. The E ring was formed by sequential depro-
tection with HCl/MeOH and cyclization with Et3N/MeOH. The
resulting intermediate was further protected with a Boc group to
give 5 for easy purification. The lactam carbonyl was then reduced
to a methylene using BMS/THF to afford 6. After the removal of
the Boc group, 13-aza-cryptopleurines (7aꢀq) were obtained
through either acylation or reductive amination (Scheme 2).
The GI50 values of 7aꢀq are listed in Table 2. Although the
13-aza analogues showed significantly reduced activity as com-
pared with that of R-cryptopleurine, some interesting results
were observed. Compound 7g, with a N-cyclopropylmethyl
substituent, was the most potent analogue against the HCT-8
cell line with a GI50 value of 0.25 μM, in addition to better
selectivity against A549 and HCT-8 versus DU145 and KB.
However, 7h, with a N-cyclopropyl substituent, one ꢀCH2ꢀ
shorter than 7g, showed dramatically reduced activity. Similar
results were observed from the comparison of 7c and 7f, as well as
antofine analogues 3f, 3a, and 3d, suggesting that the length of
the side chain on nitrogen affects the anticancer activity. It is also
interesting to note that 7g was much more potent than its R
isomer 7q, whereas 7m and 7p exhibited the reversed order of
activity, although to a less significant degree. Of all the tested
analogues in this series, compound 7a, the hydrochloride salt of
13-aza-cryptopleurine, showed considerable anticancer activity
against all four tested cell lines, indicating that 7a might be a
promising lead meriting further investigation.
’ RESULTS AND DISCUSSION
Initially, antofine analogues bearing a N atom at position C12
were designed and synthesized. The key intermediate 1 was
prepared via a procedure recently reported by our group.27 The
amino group of 1 (S or R isomer) was protected initially with a
Boc group to give 2. Then the hydroxy group was oxidized with
Py SO3 to an aldehyde, which was then converted to various
3
secondary amines through reductive amination using NaBH3CN.
After the removal of the Boc group, 3aꢀ3l were obtained through
cyclization with formaldehyde (Scheme 1).
Compounds 3aꢀ3l were then screened against four cancer
cell lines, A549 (lung), DU-145 (prostate), KB (nasopharyngeal),
and HCT-8 (colon). The screening results are shown in Table 1.
In comparison with R-antofine, all 12 compounds exhibited sub-
stantially decreased activity with an average GI50 over 1 μM. In
addition, no cell-line selectivity was observed. Compound 3f
(R = Ph) was the least potent (inactive) with an average GI50 value
In the following studies, a series of cryptopleurine analogues
(10aꢀj) with N replacement at position C12 were synthesized to
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Table 1. GI50 Values of 12-Aza-antofines 3aꢀl against Four Cancer Cell Lines
compd.
3a
R
Config.
A549 (μM)
DU145 (μM)
KB (μM)
HCT-8 (μM)
3.63 ( 0.81
-Bn
S
S
S
S
S
S
S
S
S
R
R
R
R
5.30 ( 1.02
2.00 ( 0.36
3.94 ( 0.78
6.62 ( 2.04
4.41 ( 0.97
>20
4.95 ( 1.24
6.60 ( 1.45
7.38 ( 1.55
10.20 ( 3.13
6.36 ( 1.29
>20
5.70 ( 1.52
4.40 ( 1.23
6.64 ( 1.73
10.03 ( 2.11
7.83 ( 1.59
>20
3b
-iBu
-nPr
1.82 ( 0.45
2.71 ( 0.49
7.47 ( 1.68
10.77 ( 2.44
>20
3c
3d
-(CH2)2Ph
ꢀ20ꢀOH-Et
-Ph
3e
3f
3g
-cPr
1.80 ( 0.36
0.66 ( 0.11
1.67 ( 0.37
2.68 ( 0.51
4.15 ( 0.87
0.66 ( 0.10
22 ( 7 nM
3.70 ( 0.81
2.03 ( 0.38
2.26 ( 0.47
4.62 ( 1.16
6.01 ( 1.50
1.00 ( 0.23
25 ( 5 nM
2.71 ( 0.89
1.72 ( 0.55
2.31 ( 0.65
3.41 ( 0.78
2.38 ( 0.60
0.79 ( 0.21
36 ( 8 nM
1.85 ( 0.48
1.00 ( 0.19
2.01 ( 0.54
3h
-Me
3i
-NMe2
-Bn
3j
1.87 ( 0.43 (KBvin)
10.98 ( 2.92 (KBvin)
0.66 ( 0.16
3k
-NMe2
-Me
3l
R-antofine
-
Scheme 2a
a Reagents and conditions: (a) (i) Py SO3, DMSO, Et3N, CH2Cl2; (ii) glycine methyl ester hydrochloride, Et3N, HOAc, NaBH3CN, MeOH; (b) (i)
3
HCl, MeOH; (ii) MeOH, Et3N; (iii) (Boc)2O, Et3N, CH2Cl2; (c) BMS, THF; (d) (i) TFA, CH2Cl2; (ii) RCOCl, Et3N, CH2Cl2 or RCHO, Et3N,
HOAc, NaBH3CN, MeOH.
explore their anticancer activity. Compound 2 was converted to
vinylmethyl ether 8 in two steps, oxidation followed by a Wittig
reaction using Ph3P=CH2OMe. Compound 8 was then hydro-
lyzed with Hg(OAc)2 to give aldehyde 9. The targets, 12-aza-
cryptopleurines 10aꢀj, were prepared using a strategy similar to
that described in the synthesis of compounds 3aꢀl (Scheme 3).
The GI50 values for 10aꢀj are listed in Table 3. Overall, the
results suggested that N-incorporation at position C12 of
cryptopleurine diminished the anticancer activity (lowest GI50
value was 1.12 μM for a 12-aza analogue 10j versus nM values for
cryptopleurine). Anticancer activity patterns similar to those
described above with the other compound series were observed.
Compounds with bulky or aromatic groups, such as a phenyl or
benzyl moiety (10b or 10d), exhibited lower activity (>20 μM),
as also observed in the above two series of analogues. Introducing
a polar group, such as hydroxyethyl in 10a/10i and N,N-
dimethylamino in 10f/10h, did not lead to increased activity,
although some moderate cell-line selectivity was found. The
stereochemistry of the analogues also affected the anticancer
activity (compare 10a/10i, 10c/10g, and 10f/10h), as was also
observed in the above two analogue series, although the trends
were neither significant nor consistent. Interestingly, in the
comparison of 10j and 7q (N12 vs N13 substitution), the former
compound was significantly more potent than the latter.
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Table 2. GI50 Values of 13-Aza-cryptopleurines 7aꢀq against Four Cancer Cell Lines
compd.
R
config.
A549 (μM)
DU145 (μM)
KB (μM)
HCT-8 (μM)
1.62 ( 0.56
7a
7b
7c
7d
7e
7f
H, HCl salt
-Et
S
S
S
S
S
S
S
S
S
S
S
S
S
R
R
R
R
R
1.73 ( 0.52
7.13 ( 3.86
10.93 ( 5.10
11.65 ( 1.88
14.52 ( 3.02
14.74 ( 2.49
0.79 ( 0.11
>20
2.08 ( 0.27
10.48 ( 2.51
12.19 ( 3.04
11.94 ( 2.22
16.97 ( 3.14
>20
1.79 ( 0.37
8.24 ( 1.89
11.32 ( 2.25
15.46 ( 2.78
17.52 ( 3.10
14.65 ( 2.88
6.57 ( 1.56
>20
9.45 ( 3.21
12.05 ( 2.58
13.82 ( 2.80
17.83 ( 3.63
14.03 ( 2.12
0.25 ( 0.11
13.02 ( 3.03
18.11 ( 4.91
7.41 ( 1.30
-CO2Me
-Ac
-Ms
-CH2CO2Me
-CH2cPr
-cPr
7g
7h
7i
2.94 ( 0.45
17.92 ( 5.28
15.71 ( 4.32
12.62 ( 5.61
14.66 ( 4.59
7.13 ( 1.05
>20
-Bz
15.00 ( 3.77
14.08 ( 5.49
12.18 ( 3.18
9.73 ( 1.81
>20
>20
7j
ꢀ20ꢀOH-Et
-PO(OMe)2
-Me
8.62 ( 2.44
8.09 ( 1.02
5.30 ( 0.46
16.85 ( 5.64
12.32 ( 2.15
11.25 ( 2.36
12.14 ( 2.91
>20
7k
7l
10.42 ( 2.21 (KBvin)
6.45 ( 1.03
7m
7n
7o
7p
7q
-Bn
15.24 ( 3.35 (KBvin)
11.41 ( 2.10 (KBvin)
9.96 ( 1.89 (KBvin)
7.64 ( 1.07 (KBvin)
13.44 ( 2.42 (KBvin)
1.09 ( 0.20 nM
-CONMe2
-iBu
14.08 ( 1.86
12.86 ( 2.31
16.43 ( 6.08
>20
13.12 ( 1.98
12.24 ( 4.04
11.87 ( 3.92
>20
-Bz
-CH2cPr
crypto-pleurine
1.38 ( 0.56 nM
1.59 ( 0.53 nM
1.51 ( 0.33 nM
Scheme 3a
investigating the effect of O atom incorporation at corresponding
positions. Unfortunately, introduction of the oxygen atom at
position C12 of antofine was not successful due to the instability
of the resulting analogue. However, incorporation of the O atom
into the E-ring of cryptopleurine at position C12 or C13 was
achieved, as described in Scheme 4.
Reaction of 1 with chloroacetyl chloride furnished the inter-
mediate amide, which underwent intramolecular nucleophilic
attack using NaH/THF and subsequent reduction using BMS/
THF to afford S-13-oxo-cryptopleurine 11 and its R isomer 12.
For the O replacement at position C12 of cryptopleurine, the
aldehyde of 9 was first converted to an hydroxymethyl group by
NaBH4 reduction, followed by removal of Boc and subsequent
cyclization using HCHO to give S-12-oxo-cryptopleurine 13 and
its R isomer 14 (Scheme 4).
A cytotoxicity evaluation of 11ꢀ14 against four cancer cell
lines was conducted, and the results are summarized in Table 4. It
is exciting to note that 11, with O at position C13, exhibited
potent nanomolar anticancer activity with greater selectivity
toward the HCT-8 tumor cell line (GI50 = 9 nM). In contrast,
compound 12, the R-enantiomer of 11, showed significantly
decreased activity. As for the incorporation of O at position C12,
both analogues 13 and 14 exhibited much lower potency than R-
crytopleurine with GI50 >1 μM. The slight structural variation
among 11ꢀ14 resulted in significantly different biological activ-
ity. Compound 11 might achieve a specific structural conforma-
tion that is required for interacting with the target protein leading
to the observed anticancer activity. However, further studies are
a Reagents and conditions: (a) (i) Py SO3, DMSO, Et3N, CH2Cl2; (ii)
3
Ph3P+CH2OMeClꢀ, THF, KOtBu; (b) Hg(OAc)2, THF, H2O; (c) (i)
RNH2, HOAc, NaBH3CN, MeOH; (ii) TFA, CH2Cl2; (iii) K2CO3,
MgSO4, HCHO, CH2Cl2.
Likewise, 10g was more potent than 7o, likely indicating that the
relocation of the N atom could cause a possible conformational
change, which led to a differentiation in biological activity.
Even though the above modifications of antofine and crypto-
pleurine by N incorporation in the E-ring did not provide
analogues with enhanced or at least comparable anticancer
activity to the natural alkaloids, we extended our studies by
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Table 3. GI50 Values of Analogues 10aꢀj against Four Cancer Cell Lines
compd.
R
config.
A549 (μM)
DU145 (μM)
KB (μM)
HCT-8 (μM)
10.65 ( 3.73
10a
10b
10c
10d
10e
10f
10g
10h
10i
-20-OH-Et
-Ph
S
5.68 ( 1.36
>20
1.99 ( 0.52
>20
1.59 ( 0.36
>20
S
>20
-iBu
S
1.52 ( 0.38
>20
2.30 ( 0.55
>20
5.06 ( 1.82
>20
1.36 ( 0.29
-Bn
S
>20
-cPr
S
4.30 ( 0.77
1.78 ( 0.43
5.11 ( 1.62
5.20 ( 1.02
2.48 ( 0.36
1.12 ( 0.29
1.38 ( 0.56 nM
14.33 ( 3.80
2.97 ( 0.51
1.66 ( 0.43
3.00 ( 0.65
1.46 ( 0.33
1.20 ( 0.28
1.59 ( 0.53 nM
11.47 ( 3.72
3.08 ( 0.50
1.45 ( 0.22
2.84 ( 0.56
2.22 ( 0.41
2.10 ( 0.39
1.51 ( 0.33 nM
5.73 ( 1.14
-NMe2
-iBu
S
2.37 ( 0.78
R
R
R
R
R
7.32 ( 1.90 (KBvin)
5.30 ( 1.18 (KBvin)
>20 (KBvin)
5.68 ( 1.53 (KBvin)
1.09 ( 0.20 nM
-NMe2
-20-OH-Et
-CH2cPr
10j
crypto-pleurine
Scheme 4a
against DU145 and KB (Table 5). Compound 16 was slightly more
active than 15 against DU145 and KB cell proliferation, while it was
slightly less active against A549 and HCT-8. These results suggested
that the remarkable cell-line selectivity of 15 was somewhat
decreased by introducing an oxygen atom at C13. Nevertheless,
both analogues exhibited strong potency against the HCT-8 cell line,
which indicated that phenanthroindolizidine and phenanthro-
quinolizidine derivatives with a 7-membered E-ring have a new
and interesting structural scaffold and could have a great potential
for further development as selective anticolorectal cancer agents.
In consideration of the impressive cytotoxicity, we next aimed our
study at the potential mechanism(s) of action of the representative
analogues 11 and 15, as well as R-antofine. Our preliminary cDNA
microarray data indicated that R-antofine had a profound influence
on the major replication initiation complexes ORC (origin recogni-
tion complex) and MCM (DNA replication licensing factor) in
early S phase. The DNA replication licensing factors Cdt1 (DNA
replication factor) and CDC6 (cell division control protein 6) were
also up-regulated in a dose- or time-dependent manner when
CL1ꢀ5 cells were exposed to R-antofine for 24 and 48 h, as
confirmed by RT-PCR (0.01 μg/mL, Figure 2) and Western blot
analysis (Figure 3). These data indicated that R-antofine promotes
dysregulation of DNA replication in lung cancer cells. However, it is
not clear whether the cytotoxic effect of R-antofine is directly
correlated with promoting dysregulation of DNA replication. In
general, the effects of 11 and 15 were not significant, falling in
between those produced by R-antofine and control. An in-depth
investigation on diverse cell events should be helpful for further
clarification of their mechanism of action. Obviously, the difference
in the E-ring structure is involved in these observed effects, and the
E-ring is not totally indispensable.
a Reagents and conditions: (a) (i) ClCH2COCl, Et3N, CH2Cl2; (ii)
NaH, THF, reflux; (iii) BMS, THF; (b) (i) NaBH4, MeOH; (ii) TFA,
CH2Cl2; (iii) K2CO3, MgSO4, HCHO, CH2Cl2.
needed to verify such a hypothesis, as the decreased activity of 13
and 14 could also possibly be due to molecular instability (the
hemiaminal ether E-ring).
Our study of O incorporation in antofine/cryptopleurine-type
compounds was also extended to the previously reported 7-mem-
bered E-ring analogue 15.29 Its O-containing analogue 16 was
synthesized using procedures similar to those for 11 (Scheme 5).
First, the aldehyde of 9 was reduced to a hydroxymethyl group
followed by the removal of Boc. The resulting amino intermediate
was then reacted with chloroacetyl chloride at 0 ꢀC to provide the
chloroacetamide, which was subjected to ring closure and sub-
sequent reduction of the amide carbonyl to a methylene with
BMS to give compound 16 in 24% yield over five steps.
When tested in parallel for cytotoxicity, both 15 and 16 were
more active against A549 and HCT-8 cancer cell lines than
Compound 11 was then selected for an in vivo study in mice
against HT-29 human colorectal adenocarcinoma xenograft.
Mice in group 1 received the vehicle and served as the control
for all treatment groups. The median time to end point (TTE)
for mice in the control group was 16.3 days with a range of
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Table 4. GI50 Values of Analogues 11ꢀ14 against Four Cancer Cell Lines
compd.
config.
A549
DU145
KB
HCT-8
11
12
13
14
S
23 ( 5 nM
67 ( 10 nM
37 ( 9 nM
9 ( 5 nM
R
S
13.95 ( 4.74 μM
1.79 ( 0.27 μM
1.37 ( 0.29 μM
1.38 ( 0.56 nM
7.05 ( 1.25 μM
1.82 ( 0.31 μM
1.29 ( 0.36 μM
1.59 ( 0.53 nM
6.68 ( 1.52 μM
1.66 ( 0.33 μM
1.10 ( 0.28 μM
1.51 ( 0.33 nM
3.45 ( 0.56 μM
1.45 ( 0.24 μM
1.26 ( 0.20 μM
1.09 ( 0.20 nM
R
R
crypto-pleurine
Scheme 5a
a Reagents and conditions: (a) (i) NaBH4, MeOH; (ii) TFA, CH2Cl2; (iii) ClCH2COCl, Et3N, CH2Cl2, 0 ꢀC; (iv) NaH, THF, reflux; (v) BMS, THF,
24% for 5 steps.
Table 5. GI50 Values of Compounds 15 and 16
R-cryptopleurine was reduced by 30-fold through our modifica-
tion, as shown in Figure 6 (GI50 320 nM vs 11 nM). Such
improved selectivity against cancer cells over normal cells demon-
strates that our newly synthesized analogues can potentially
reduce unwanted side effects in vivo. In summary, compound
11 exhibited potent in vitro anticancer activity with improved cell
line selectivity and moderate antitumor activity against HT-29
human colorectal adenocarcinoma in male nude-athymic mice at a
dose of 20 mg/kg without causing obvious toxicity.
compd. config. A549 (nM) DU145 (nM) KB (nM) HCT-8 (nM)
15
16
R
S
25 ( 5
41 ( 9
179 ( 36
119 ( 33
102 ( 19
68 ( 10
10 ( 3
20 ( 6
13.8ꢀ21.9 days. Compound 11, i.v. at 20 mg/kg (qd to end),
produced a median TTE of 21.3 days and caused 1.1% maximum
group body weight loss on day 12, indicating statistically significant
antitumor activity (P < 0.05) and no obvious toxicity. When
dosed i.v./i.p. at 20 mg/kg (bid to end), 11 produced a median
TTE of 20.9 days and caused 7.2% maximum group body weight
loss occurring on day 19. One tumor remained in the study on
day 29, and one treatment-related (TR) death occurred on day
20 (Figures 4 and 5). When dosing frequency was increased, no
improved efficacy was observed. Interestingly, a similar phenomenon
was also reported in previous studies. For instance, the effectiveness
of phenanthroindolizidine analogues with C14-OH was found to
be highly schedule dependent, e.g., the therapeutic index of
DCB-3503 may be lost if given daily instead of every three days.34
Therefore, a more rationally designed dosing regimen is being
investigated in order for the compounds to exert their best tumor
inhibitory effects and meanwhile reduce toxicity that could result
from unnecessary repetitive dosing. Moreover, we tested compound
11 against human umbilical vein endothelial cells (HUVECs,
normal cells), and the results indicated that the cytotoxicity of
’ CONCLUSIONS
New synthetic antofine and cryptopleurine analogues with
varied E-ring size or heteroatom incorporation were designed
and prepared. Two analogues, 11 and 16, were the most active
compounds against tested cancer cell lines, with interestingly
improved selectivity against HCT-8 cell growth. Mechanistic
studies indicated that R-antofine is capable of promoting dysre-
gulation of DNA replication in early S phase, whereas no similar
results were found for compounds 11 and 15. It is still unclear as
to how this activity correlates with the potent cytotoxicity of
antofine, and further investigation is ongoing. In our in vivo
antitumor study, we found that 11 had improved activity on
HUVECs and moderate antitumor effect on human HT-29
adenocarcinoma xenograft in mice. However, further studies in
other tumor models are warranted to find out which type of
tumor affords the most sensitivity and what treatment protocol
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provides the best therapeutic index. Moreover, compound 16
appears to be an interesting analogue that necessitates in-depth
investigation, and results will be reported in due course. Addi-
tionally, CNS toxicity will also be explored in the future.
300 MHz Gemini or a Varian Inova (400 MHz) NMR spectrometer
with TMS as the internal standard. The solvent used was CDCl3 unless
otherwise indicated. Mass spectra were recorded on a Shimazu-2010
LC/MS/MS instrument equipped with a TurboIonsSpray ion source.
All final target compounds were characterized and determined as at least
>95% pure by analytical HPLC.
’ EXPERIMENTAL SECTION
(S)-N-Boc-(6,7,10-trimethoxy-1,2,3,4-tetrahydrodibenzo-
[f,h]isoquinolin-3-yl)methanol (2). (Boc)2O (3.14 g, 14.40 mmol)
was added to the amino-alcohol (4.24 g, 12 mmol) in 100 mL of CH2Cl2
and Et3N (6 mL), and the mixture was stirred for 2 h. HCl (50 mL, 1 N)
was added, and the organic layer was separated, washed with sat.
NaHCO3 and brine, and dried over MgSO4. Column chromatography
eluting with CH2Cl2/MeOH gave 5.20 g of compound 2 as a white solid.
All chemicals were used as purchased. Melting points were measured
using a Fisher Johns melting apparatus without correction. Proton
nuclear magnetic resonance (1H NMR) spectra were measured on a
1
Yield: 95.6%; mp 105ꢀ107 ꢀC; [R]23D= 85.5ꢀ (c 0.42, CHCl3). H
NMR (300 MHz, CDCl3): δ 7.89 (s, 2H), 7.81 (d, J = 9.0 Hz, 1H), 7.28
(s, 1H), 7.22 (d, J = 8.7 Hz, 1H), 5.29 (br s, 1H), 4.84 (m, 1H), 4.61 (br s,
1H), 4.09 (s, 3H), 4.05 (s, 3H), 4.01 (s, 3H), 3.73ꢀ3.62 (m, 2H), 3.27
(dd, J = 16.5 Hz, J = 6.3 Hz, 1H), 3.18 (t, J = 16.5 Hz, 1H), 1.55 (s, 9H).
13C NMR (75 MHz, CDCl3): δ 157.9, 156.2, 149.7, 148.8, 130.4, 126.7,
124.0, 123.9, 123.8, 123.4, 122.8, 115.2, 105.1, 104.2, 104.0, 80.6, 62.7,
56.2, 56.0, 55.7, 50.6, 41.0, 28.6 (3 ꢁ C), 26.5. ESI-HRMS ([M + H]+)
calcd for C26H32NO6 454.2230; found, 454.2246.
General Procedures for the Synthesis of 12N-Substituted-
12-aza-antofine (3aꢀl). The aldehyde was obtained using the same
procedure as that for compound 8. Various amines were reacted with the
aldehyde in MeOH, followed by the addition of HOAc and NaBH3CN,
which was stirred at r.t. for 4 h. Saturated NaHCO3 was added to quench
the reaction, and CH2Cl2 was used for extraction. The residue was
quickly purified through a column, and TFA was used to remove the Boc
group. Then TFA was removed by evaporation, and the residue was
dissolved in CH2Cl2 (10 mL), to which K2CO3 (100 mg) and MgSO4
(0.5 g) were added, followed by HCHO (37%, 0.1 mL). The mixture was
stirred at r.t. overnight. Chromatography gave light yellow to white
solids. Yield: 40%ꢀ80%.
(S)-tert-Butyl-6,7,10-trimethoxy-3-((2-methoxy-2-oxo-
ethylamino)methyl)-3,4-dihydrodibenzo[f,h]isoquinoline-
2(1H)-carboxylate (4). The Boc-aldehyde was obtained using the
same procedure as that used for compound 8. Glycine methyl ester
Figure 2. RT-PCR results of R-antofine, 11, and 15 at 0.01 μg/mL on
major DNA replication complexes in CL1ꢀ5 cells at 24 and 48 h.
Figure 3. Western blot analysis of R-antofine, 11, and 15 on major DNA replication complexes in CL1ꢀ5 cells at 24 and 48 h.
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Figure 4. Individual animal body weight change for control and treatment groups.
hydrochloride (314 mg, 2.5 mmol) and Et3N (0.35 mL, 2.5 mmol) were
(S)-13N-Boc-13-aza-cryptopleurine (6). To a solution of the
added to the aldehyde in MeOH (30 mL), which was stirred for 0.5 h. Then
HOAc (0.70 mL, 12 mmol) and NaBH3CN (165 mg, 2.5 mmol) were
added. The mixture was stirred for 2 h until all the aldehyde disappeared.
Saturated NaHCO3 was used to quench the reaction, and CH2Cl2 was used
for extraction, and washed with brine, dried over MgSO4. Chromatography
gave 635 mg of compound 4 as a white solid. Yield: 61% over two steps. mp
78ꢀ80 ꢀC; [R]23D= 91.6ꢀ (c 0.37, CHCl3). 1H NMR (400 MHz, CDCl3):
δ7.92 (s, 1H), 7.91 (d, J= 2.4 Hz, 1H), 7.87 (brs, 1H), 7.44ꢀ7.42 (m, 2H),
7.29 (s, 1H), 7.25ꢀ7.23 (m, 1H), 5.40ꢀ5.29 (m, 1H), 4.92ꢀ4.78 (m, 1H),
4.56 (m, 1H), 4.11 (s, 3H), 4.05 (s, 3H), 4.02 (s, 3H), 3.66 (s, 3H), 3.47ꢀ
3.36 (m, 2H), 3.27 (dd, J = 16.4 Hz, J = 6.4 Hz, 1H), 3.14 (d, J = 16.4 Hz,
1H), 2.81 (dd, J = 12.0 Hz, J = 8.8 Hz, 1H), 2.64 (dd, J = 12.0 Hz, J = 6.4 Hz,
1H), 1.54 (s, 9H). ESI MS m/z 525.20 (M + H)+.
amide (456 mg, 0.93 mmol) in THF (30 mL) was added BMS (2 M in
THF, 2.79 mL), which was stirred at r.t. overnight. Five milliliters of
MeOH was added, and the mixture was refluxed for 1 h. Chromatog-
raphy afforded 400 mg of 6 as a white solid. Yield: 90%. mp 116ꢀ118 ꢀC;
1
[R]23D= ꢀ182.5ꢀ (c 0.28, CHCl3). H NMR (400 MHz, CDCl3): δ
7.88ꢀ7.87 (m, 2H), 7.76 (d, J = 8.8 Hz, 1H), 7.20 (s, 1H), 7.19 (dd, J =
8.8 Hz, J = 2.8 Hz, 1H), 4.44 (d, J = 15.2 Hz, 1H), 4.29 (m, 1H), 4.11 (m,
1H), 4.09 (s, 3H), 4.05 (s, 3H), 4.00 (s, 3H), 3.66 (d, J = 15.2 Hz, 1H),
3.15 (m, 2H), 3.04 (dd, J = 16.4 Hz, J = 3.2 Hz, 1H), 2.87 (m, 1H), 2.80
(dd, J = 16.4 Hz, J = 11.8 Hz, 1H), 2.57ꢀ2.52 (m, 1H), 2.45 (dt, J = 12.0
Hz, 3.2 Hz, 1H). ESI MS m/z 479.10 (M + H)+.
General Procedures for the Synthesis of 13N-Substituted-
13-aza-cryptopleurine (7aꢀq). (a) 13N-Boc-13-aza-cryptopleur-
ine 6 was stirred in HCl/MeOH for 2 h before the solvent was removed
in vacuo. The solid was collected and washed sequentially with cold
MeOH and ether. Then the solid was dissolved in 10 mL of anhydrous
CH2Cl2 and Et3N (0.1 mL), to which RCOCl was added at 0 ꢀC. The
mixture was stirred at r.t. for 4 h before 1 N HCl was added. After routine
workup, chromatography afforded from light orange to white solids.
Yield: 70%ꢀ80%. (b) The same procedures (reductive amination) as
those for compound 4 were used. Yield: 50%- 80%.
(S)-13N-Boc-11-oxo-13-aza-cryptopleurine (5). The ester
(635 mg, 1.21 mmol) was dissolved in HCl (1.25 M in methanol,
15 mL), which was stirred at 50 ꢀC for 1 h. The solvent was removed.
Thirty milliliters of MeOH and Et3N (0.3 mL) was added. The mixture
was stirred at r.t. for 2 h. After normal workup, chromatography afforded
404 mg of ring-closed intermediate as a white solid. Using a procedure
similar to that for compound 2, the intermediate was protected by
(Boc)2O to give 456 mg of 5 as a white solid. Yield: 76% over three steps.
1
mp 125ꢀ127 ꢀC; [R]23D= ꢀ202.8ꢀ (c 0.50, CHCl3). H NMR (400
(S,E/Z)-6,7,10-Trimethoxy-3-(2-methoxyvinyl)-1,2,3,4-tetra-
hydrodibenzo[f,h]isoquinoline (8). N-Boc-alcohol 2 (1.812 g,
4 mmol) was dissolved in CH2Cl2 (50 mL) and Et3N (1.95 mL, 14 mmol)
MHz, CDCl3): δ 7.89 (s, 1H), 7.88 (d, J = 2.8 Hz, 1H), 7.87 (d, J = 9.2
Hz, 1H), 7.23 (dd, J = 9.2 Hz, J = 2.8 Hz, 1H), 7.19 (s, 1H), 5.88 (d, J =
17.2 Hz, 1H), 4.49 (d, J = 17.2 Hz, 1H), 4.40 (d, J = 18.0 Hz, 1H), 4.10
(s, 4H), 4.05 (s, 4H), 4.01 (s, 3H), 3.87ꢀ3.77 (m, 2H), 3.16 (m, 2H),
1.51 (s, 9H). ESI MS m/z 493.10 (M + H)+, 985.20 (2M + H)+.
at 0 ꢀC, to which Py SO3 (2.23 g, 14 mmol) in 10 mL of DMSO was
3
added dropwise. The ice bath was then removed, and the mixture was
stirred at r.t. until 2 disappeared by TLC. HCl (1 N) was added, and the
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Figure 5. In vivo antitumor activity of compound 11.
Figure 6. Comparison of compound 11 and R-cryptopleurine against human umbilical vein endothelial cells (HUVECs).
organic layer was then separated and washed with sat. NaHCO3, brine,
and dried over MgSO4. In another flask, Ph3P+CH2OMeClꢀ (2.74 g, 8
mmol) was added to KOtBu (875 mg, 7.8 mmol) at 0 ꢀC under N2,
which was stirred for 0.5 h. The aldehyde in 20 mL of CH2Cl2 was added
dropwise, followed by the removal of the ice bath. The reaction was kept
for 2ꢀ3 h before sat. NH4Cl was poured into the mixture. CH2Cl2 was
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used for extraction, and the organic layers were washed with sat.
NaHCO3 and brine, dried over MgSO4. Chromatography afforded 1.5
g of compound 8 as a light yellow solid. Yield: 78% (Z/E = 1/2.5) for two
steps. mp 150ꢀ152 ꢀC; [R]23D= 108.1ꢀ (c 0.59, CHCl3). 1H NMR (400
MHz, CDCl3, E/Z = 2.5:1): δ 7.94ꢀ7.91 (m, 2H), 7.88 (d, J = 9.2 Hz,
1H), 7.29 (s, 1H), 7.26ꢀ7.22 (m, 1H), 6.30 (d, J = 12.8 Hz, 0.68H, E
isomer), 5.86 (dd, J = 6.4 Hz, J = 1.6 Hz, 0.28 Hz, Z isomer), 5.72 (m,
0.31H, Z isomer), 5.30ꢀ5.25 (d, J = 16.8 Hz, 1H+0.67H, E isomer), 4.80
(dd, J = 12.8 Hz, J = 8.4 Hz, 0.71 H, E isomer), 4.59 (d, J = 17.2 Hz, 1H),
4.42 (dd, J = 8.0 Hz, J = 6.4 Hz, 0.29H, Z isomer), 4.12ꢀ4.11 (m, 3H),
4.05ꢀ4.04 (m, 3H), 4.03ꢀ4.02 (m, 3H), 3.62 (s, 0.71H, Z isomer),
3.44ꢀ3.32 (m, 1H), 3.36 (s, 2H, E isomer), 3.13 (d, J = 16.0 Hz, 1H),
1.55 (s, 9H). ESI MS m/z 480.05 (M + H)+.
(S)-2-(6,7,10-Trimethoxy-1,2,3,4-tetrahydrodibenzo[f,h]iso-
quinolin-3-yl)acetaldehyde (9). Compound 8 (1.5 g, 3.12 mmol)
was dissolved in THF (50 mL) and H2O (5 mL), to which Hg(OAc)2
(3.0 g, 9.36 mmol) was added at 0 ꢀC. Then the ice bath was removed,
and the mixture was stirred overnight. Freshly prepared sat. KI (50 mL)
was added dropwise at 0 ꢀC for 10 min, and CH2Cl2 was used for extrac-
tion. The organic layers were collected and washed with brine, dried over
MgSO4. Chromatography furnished 1.09 g of compound 9 as a light yellow
foam. Yield: 75%. mp 98ꢀ100 ꢀC; [R]23D= 94.4ꢀ (c 0.68, CHCl3). 1H
NMR (400 MHz, CDCl3): δ 9.79 (s, 1H), 7.93 (s, 1H), 7.91 (d, J = 2.4
Hz, 1H), 7.85 (brs, 1H), 7.26ꢀ7.24 (m, 2H), 5.38ꢀ5.29 (m, 2H), 4.58
(d, J = 16.0 Hz, 1H), 4.11 (s, 3H), 4.05 (s, 3H), 4.02 (s, 3H), 3.38 (dd, J =
16.0 Hz, J = 6.4 Hz, 1H), 3.12 (d, J = 16.4 Hz, 1H), 2.70ꢀ2.59 (m, 2H),
1.53 (s, 9H). ESI MS m/z 466.10 (M + H)+, 488.15 (M + Na)+.
General Procedures for the Synthesis of 12N-Substituted-
12-aza-cryptopleurine (10aꢀj). Similar procedures as those used
for compound 3aꢀl were used. Yield: 50%ꢀ70%.
workup, 80 mg of white solid was obtained, which was used without
further purification. The residue was dissolved in CH2Cl2 (10 mL), to
which MgSO4, K2CO3, and HCHO were sequentially added. The mixture
was stirred overnight. After workup, chromatography gave 32 mg of 13 as
a white solid. Yield: 40% for two steps; light yellow solid; mp
1
195ꢀ197 ꢀC; [R]23D= ꢀ56.8ꢀ (c 0.60, CHCl3). H NMR (300 MHz,
CDCl3): δ7.89ꢀ7.88 (m, 2H), 7.75 (d, J = 9.3 Hz, 1H), 7.22 (s, 1H), 7.19
(dd, J = 9.0 Hz, J = 2.7 Hz, 1H), 4.79 (d, J = 8.4 Hz, 1H), 4.41 (d, J = 15.3
Hz, 1H), 4.19 (dd, J = 11.1 Hz, J = 5.1 Hz, 1H), 4.10 (s, 3H), 4.05 (s, 3H),
4.03 (m, 1H), 4.01 (s, 3H), 3.75ꢀ3.65 (m, 2H), 3.17 (dd, J = 15.9 Hz, J =
3.6 Hz, 1H), 2.95ꢀ2.80 (m, 2H), 2.06ꢀ1.92 (m, 1H), 1.74 (m, 1H); ESI
MS m/z 380.05 (M + H)+. For 14: mp 191ꢀ192 ꢀC; [R]23D= 51.2ꢀ
(c0.60, CHCl3). 1H NMR (300 MHz, CDCl3):δ7.87ꢀ7.86 (m, 2H), 7.73
(d, J = 9.0 Hz, 1H), 7.20 (s, 1H), 7.18 (dd, J = 9.6 Hz, J = 3.0 Hz, 1H), 4.77
(d, J = 8.1 Hz, 1H), 4.38 (d, J = 15.3 Hz, 1H), 4.18 (dd, J = 11.1 Hz, J = 4.8
Hz, 1H), 4.09 (s, 3H), 4.04 (s, 3H), 3.99 (s, 3H), 3.98 (d, J = 8.1 Hz, 1H),
3.73ꢀ3.61 (m, 2H), 3.13 (dd, J = 15.9 Hz, J = 3.6 Hz, 1H), 2.91ꢀ2.72
(m, 2H), 2.02ꢀ1.90 (m, 1H), 1.72ꢀ1.67 (m, 1H). 13C NMR (75 MHz,
CDCl3): δ 157.7, 149.6, 148.5, 130.3, 126.6, 124.7, 124.2, 123.8, 123.7,
123.6, 115.0, 104.9, 104.5, 103.9, 86.5, 68.1, 56.1, 56.0, 55.7, 55.4, 48.0,
33.5, 31.3. ESI MS m/z 380.05 (M + H)+.
Compound 16. Aldehyde 9 (140 mg, 0.30 mmol) was dissolved in
MeOH, to which NaBH4 (76 mg, 2 mmol) was added. The resulting
mixture was stirred at r.t. for 1 h before sat. NaHCO3 was added. The
mixture was extracted with CH2Cl2, and the organic layers were
combined, washed with brine, and dried over Na2SO4. Then the solvent
was removed under reduced pressure and redissolved in TFA/CH2Cl2
(1:1, 10 mL), which was stirred for 0.5 h. The mixture was concentrated
to remove TFA. The residue was subject to treatment with chloroacetyl
chloride, and the rest of the synthesis was similar to that of compound
11. After chromatography, 37 mg of 16 was obtained as a light yellow
solid in 24% yield over five steps. mp 183ꢀ185 ꢀC; [R]23D= ꢀ114.7ꢀ (c
0.19, CHCl3). 1H NMR (400 MHz, CDCl3): δ 7.90 (s, 1H), 7.88 (d, J =
2.4 Hz, 1H), 7.75 (d, J = 9.2 Hz, 1H), 7.24 (s, 1H), 7.19 (dd, J = 9.2 Hz, J
= 2.4 Hz, 1H), 4.43 (d, J = 15.2 Hz, 1H), 4.10 (s, 3H), 4.05 (m, 4H), 4.01
(m, 4H), 3.98ꢀ3.90 (m, 3H), 3.20ꢀ3.13 (m, 1H), 3.08ꢀ3.01 (m, 3H),
2.96ꢀ2.91 (m, 1H), 2.25ꢀ2.19 (m, 1H), 2.17ꢀ2.11 (m, 1H). 13C NMR
(100 MHz, CDCl3): δ 157.7, 149.6, 148.6, 130.3, 126.9, 126.6, 125.4,
124.2, 123.9, 123.7, 115.1, 104.9, 104.1, 104.0, 69.9, 66.5, 59.0, 57.6, 57.0,
56.2, 56.1, 55.7, 36.7, 35.5. ESI MS m/z 394.10 (M + H)+.
(S)-13-Oxa-cryptopleurine (11) and (R)-13-Oxa-crypto-
pleurine (12). Compound 2 (177 mg, 0.5 mmol) was suspended in
dry CH2Cl2 (15 mL) and Et3N (0.14 mL, 1.00 mmol) at 0 ꢀC, to which
chloroacetyl chloride (40 μL, 0.50 mmol) in 1 mL of CH2Cl2 was slowly
added dropwise. The mixture was stirred at 0 ꢀC for 5 h before 1 N HCl
was added. The organic layer was separated and washed with sat.
NaHCO3 and brine, dried over MgSO4. CH2Cl2 was removed, and
the residue was dissolved in anhydrous THF (5 mL), to which NaH (2.1
equiv) was added at r.t. followed by reflux for 2 h. Saturated NH4Cl was
added, and CH2Cl2 was used for extraction. After workup, the organic
layer was dried over MgSO4. Then the residue was dissolved in 10 mL of
anhydrous THF, and BMS (1.50 mL, 6 mmol) was added, which was
stirred at r.t. overnight. MeOH (5 mL) was added, and the mixture was
refluxed for 1 h. Chromatography gave 73 mg of 11 as a white solid. Yield:
38.5% for three steps; white solid; mp 199ꢀ201 ꢀC; [R]23D= ꢀ134.6ꢀ
(c 0.52, CHCl3). 1H NMR (300 MHz, CDCl3): δ 7.86ꢀ7.85 (m, 2H),
7.74 (d, J = 9.0 Hz, 1H), 7.18 (dd, J = 9.0 Hz, J = 2.4 Hz, 1H), 7.14 (s,
1H), 4.39 (d, J = 15.6 Hz, 1H), 4.12 (m, 2H), 4.08 (s, 3H), 4.03 (s, 3H),
4.00 (s, 3H), 3.91ꢀ3.83 (m, 1H), 3.66 (d, J = 15.6 Hz, 1H), 3.51 (dd, J =
11.1 Hz, J = 9.0 Hz, 1H), 3.06 (d, J = 11.7 Hz, 1H), 2.91ꢀ2.87 (m, 1H),
2.76ꢀ2.59 (m, 3H). ESI MS m/z 380.05 (M + H)+. For 12: mp
’ ASSOCIATED CONTENT
S
Supporting Information. Compound data for 3aꢀl,
b
7aꢀq, and 10aꢀj, biological studies, and HPLC analysis of final
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
1
196ꢀ198 ꢀC; [R]23D= 139.6ꢀ (c 0.26, CHCl3). H NMR (300 MHz,
*(Q.S.) Phone: 919-843-6325. Fax: 919-966-3893. E-mail:
qshi1@email.unc.edu. (P.-C.Y.) Phone: 886-2-2356-2185. Fax:
886-2-2322-4793. E-mail: pcyang@ntu.edu.tw. (K.-H.L.) Phone:
919-962-0066. Fax: 919-966-3893. E-mail: khlee@unc.edu.
CDCl3): δ 7.90ꢀ7.89 (m, 2H), 7.78 (d, J = 9.0 Hz, 1H), 7.20 (dd, J = 9.0
Hz, J = 2.7 Hz, 1H), 7.20 (s, 1H), 4.44 (d, J = 15.6 Hz, 1H), 4.17ꢀ4.08
(m, 1H), 4.10 (s, 3H), 4.05 (s, 3H), 4.01 (s, 3H), 4.04ꢀ4.00 (m, 1H),
3.92ꢀ3.84 (m, 1H), 3.72 (d, J = 15.3 Hz, 1H), 3.54 (dd, J = 11.1 Hz, J =
9.0 Hz, 1H), 3.09 (d, J = 11.4 Hz, 1H), 2.98ꢀ2.94 (m, 1H), 2.77ꢀ2.61
(m, 3H). 13C NMR (75 MHz, CDCl3): δ 157.5, 149.4, 148.4, 130.2,
126.4, 125.1, 124.1, 123.6, 123.4, 123.3, 115.0, 104.7, 103.8, 103.6, 72.3, 67.4,
56.1, 56.0, 55.9, 55.5, 55.1, 54.4, 28.5. ESI MS m/z 380.05 (M + H)+.
(S)-12-Oxa-cryptopleurine (13) and (R)-12-Oxa-crypto-
pleurine (14). Aldehyde 9 (100 mg, 0.21 mmol) was dissolved in
MeOH, to which NaBH4 (19 mg, 0.50 mmol) was added in one portion.
The mixture was stirred for 1 h, and sat. NaHCO3 was added. After normal
’ ACKNOWLEDGMENT
This study was supported by grant CA17625-32 from the National
Cancer Institute awarded to K.H.L. and grant DOH98-TD-G-
111-007 from the National Research Program for Genomic
Medicine awarded to P.C.Y. This study was also supported in
part by the Department of Health Cancer Research Center of
Excellence (DOH-100-TD-C-111-05).
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’ ABBREVIATIONS USED
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(18) Cai, X. F.; Jin, X.; Lee, D.; Yang, Y. T.; Lee, K.; Hong, Y.-S.; Lee,
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MOA, mechanism of action; NCI, National Cancer Institute;
HIF-1, hypoxia-inducible factor 1; TS, thymidylate synthase;
DHFR, dihydrofolate reductase; AP-1, activator protein-1; CRE,
cAMP response element; CDK, cyclin-dependent kinase; CNS,
central neural system; SAR, structureꢀactivity relationship;
BBB, bloodꢀbrain barrier; PSA, polar surface area; ORC, origin
recognition complex; CDC6, cell division control protein 6; RT-
PCR, reverse transcription polymerase chain reaction; HUVECs,
human umbilical vein endothelial cells
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