Synthesis and cytotoxic activities of β-carboline amino acid ester conjugates

Article abstract of DOI:10.1016/j.bmc.2006.06.021

β-Carboline represents a class of compounds with potent anti-tumor activity by intercalating with DNA. To further enhance the cytotoxic potency and bioavailability of β-carboline, a series of novel β-carboline amino acid ester conjugates were designed and synthesized, and the cytotoxic activities of these compounds were tested using a panel of human tumor cell lines. In addition, the membrane permeability of these compounds was evaluated in vitro using a Caco-2 cell monolayer model. The β-carboline amino acid ester conjugates demonstrated improved cytotoxic activity compared to the parental β-carbolines. In particular, the Lys/Arg conjugates were the most potent analogs with an IC₅₀ value of 4 and 1 μM against human cervical carcinoma cells. The low interaction energy of Arg conjugate based on molecular modeling may contribute to its enhanced cytotoxicity. Taken together, this study provided new insights into structure-activity relationships in the β-carboline amino acid ester conjugates and identified the β-carboline Lys/Arg conjugates as promising lead compounds for further in vivo biological and molecular evaluation.

Full text of DOI:10.1016/j.bmc.2006.06.021

Bioorganic & Medicinal Chemistry 14 (2006) 6998–7010
Synthesis and cytotoxic activities of b-carboline amino acid
ester conjugates
b
a
b
b
d
Ming Zhao, Lanrong Bi, Wei Wang, Chao Wang, Mich e` le Baudy-Floc’h,
c,
a,
*
*
Jingfang Ju and Shiqi Peng
a
College of Pharmaceutical Sciences, Capital University of Medical Sciences, Beijing, 100054, PR China
b
College of Pharmaceutical Sciences, Peking University, Beijing 100083, PR China
Mitchell Cancer Institute, University of South Alabama, 307 N. University Blvd, Mobile, AL 36688-0002, USA
c
d
UMR 6226 CNRS, Groupe ‘‘Ciblage et Autoassemblages Fonctionnels’’, Universit e´ de Rennes I, 263 Av. du G e´ n e´ ral Leclerc,
F-35042 Rennes Cedex, France
Received 8 May 2006; revised 31 May 2006; accepted 8 June 2006
Available online 27 June 2006
Abstract—b-Carboline represents a class of compounds with potent anti-tumor activity by intercalating with DNA. To further
enhance the cytotoxic potency and bioavailability of b-carboline, a series of novel b-carboline amino acid ester conjugates were
designed and synthesized, and the cytotoxic activities of these compounds were tested using a panel of human tumor cell lines.
In addition, the membrane permeability of these compounds was evaluated in vitro using a Caco-2 cell monolayer model. The
b-carboline amino acid ester conjugates demonstrated improved cytotoxic activity compared to the parental b-carbolines. In par-
ticular, the Lys/Arg conjugates were the most potent analogs with an IC50 value of 4 and 1 lM against human cervical carcinoma
cells. The low interaction energy of Arg conjugate based on molecular modeling may contribute to its enhanced cytotoxicity. Taken
together, this study provided new insights into structure–activity relationships in the b-carboline amino acid ester conjugates and
identified the b-carboline Lys/Arg conjugates as promising lead compounds for further in vivo biological and molecular evaluation.
ꢀ
2006 Elsevier Ltd. All rights reserved.
1. Introduction
aromatic residue capable of stacking between the
,3
2
DNA pairs at the intercalation sites.
Intercalation
The discovery of new compounds with potent anti-
tumor activity is one of the most important goals in
medicinal chemistry. One interesting group of chemo-
therapeutic agents used in cancer therapy comprises
molecules, such as cisplatin and CPT-11, target directly
causes conformational changes in the double helix,
accordingly, DNA replication, transcription, and repair
4
are altered. As a consequence, they interfere with these
functions resulting in killing of fast growing cells to cre-
5
ate a therapeutic window. Therefore, potential applica-
tions of DNA intercalators can be directed toward anti-
1
to DNA. During the past decades, numerous studies
6
–8
have demonstrated DNA recognizing molecules that
act as anticancer agents, including groove binders, alkyl-
ating and intercalator compounds. Among these, DNA
intercalators represent one of the most important classes
of anticancer drugs in clinical oncology, and a few rep-
resentatives (anthracyclines, acridines, and anthraqui-
nones) are routinely used in the clinic for the
cancer drugs.
Recently, a number of compounds that intercalate with
DNA have been synthesized and tested for their antitu-
9
–16
mor activities.
For example, some b-carboline alka-
loids such as harmine and its derivatives were highly
1
7–23
cytotoxic against human tumor cell lines.
The fur-
1
treatment of cancers. DNA intercalating agents are ap-
plied to those compounds having a planar polycyclic
ther mechanistic studies indicated that the b-carbolines
could intercalate into the DNA helix and inhibit DNA
2
4–28
topoisomerase I and II to cause DNA damage.
The studies implicated a direct correlation between
2
4–28
DNA intercalating ability and cytotoxicity.
Keywords: Beta-carboline; Cytotoxicity; Anti-cancer.
*
Corresponding authors. Tel.: +251 460 7393; fax: +251 460 6994
J.J.); tel.: +86 10 8391 1528; fax: +86 10 8391 1528 (S.P.); e-mail
addresses: jju@usouthal.edu; sqpeng@mail.bjum.edu.cn
(
As part of our ongoing efforts to discover natural prod-
2
9,30
ucts with potent anti-tumor activity,
we recently
0
968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.06.021

M. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 6998–7010
6999
designed and synthesized a series of b-carboline amino
acid ester conjugates. Amino acids are attractive sub-
strates because they are not only the fundamental build-
ing blocks of biological systems and many natural
products, they also possess structurally diverse side
chains. It is well known that many amino acids with
functional side chains are capable of making base-specif-
tested against a panel of human tumor cell lines. In addi-
tion, the structure–activity relationship studies of substi-
tution preferences for enhanced cytotoxic activity have
been elucidated. Furthermore, the membrane permeabil-
ity of the derivatives was evaluated using a Caco-2 cell
monolayer experiment.
3
1
ic contacts with more than one type of DNA bases.
With this in mind, we introduced a series of amino acids
containing either nonpolar, acidic, basic, or aromatic
sides, various functionalities with absolute stereochemis-
try into the b-carbolines’ core. We postulated that the
conjugation of amino acids with the b-carbolines could
modulate the DNA intercalating capability and conse-
quently improve their biological activity through the
possible synergetic interactions between DNA with the
structurally diverse amino acid side chains and b-carbo-
lines. It was anticipated that the introduction of amino
acid side chains would provide extra structural contribu-
tion to enhance b-carbolines’ interaction capability with
DNA.
2. Results and discussion
2.1. Synthesis of b-carboline derivatives
The synthesis of b-carboline derivatives was accom-
plished by adopting the Pictet–Spengler cyclization,
which involves the acid-catalyzed cyclocondensation of
b-arylethylamine with an aldehyde or ketone. The syn-
thetic route is outlined in Schemes 1–3. Starting from
the optically active L-tryptophane, after esterification
of the carboxylic moiety with MeOH/SOCl , the result-
2
ing L-tryptophane methyl ester was then subjected to
Pictet–Spengler cyclization with aldehyde to yield methyl
1-methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylate 2
Another reason for the design of amino acid conjugates
of b-carbolines was to improve their bioavailability.
Anticancer drug efficiency is often limited by poor bio-
availability. In general, the bioavailability of the antitu-
mor agents not only affects the absorption and
transportation of the antitumor agents, but also affects
the cytotoxic potencies of the antitumor agents. Thus,
an attractive approach to improve and optimize the
pharmacologic profiles of antitumor agents is to increase
their oral bioavailability. Previous studies on the phar-
macokinetic disposition revealed that the oral bioavail-
in 47% yield. The cyclization of the chiral substrate
was carried out using sulfuric acid as catalyst, 2:1
trans/cis (2a/2b) diastereomers of tetrahydro-b-carboline
derivatives were isolated by chromatography.
To control the chirality in the Pictet–Spengler reaction
3
for the enantiospecific synthesis of indole alkaloids,
0a,b
the effect of substitution on the stereospecificity was
investigated. When L-tryptophane was directly em-
ployed as the substrate, the Pictet–Spengler reaction
provided trans/cis diasetereomers 1a/1b in a ratio of
16:1 (85% yield). After the esterification, trans diastereo-
mer of the carboxylate methyl ester 2a was generated as
the predominant product (trans/cis 16:1). The increase in
stereoselectivity in favor of the trans diastereomer was
notable, which was probably due to the influence of
the substituents on the stereospecificity.
3
2
ability of b-carbolines was relatively low (<20%).
Considering amino acid conjugates might target the gas-
trointestinal transporters involved in the absorption of
amino acids and small peptides therefore result in im-
3
3–36
proved oral bioavailability,
amino acids were engi-
neered with b-carbolines to improve the bioavailability
and biological activity. In this regard, we expect that
amino acid conjugate approach would be a useful strat-
egy for improving the intestinal absorption of drug with
The conversion of the 1,2,3,4-tetrahydro-b-carboline
derivative 2 to the corresponding b-carboline derivative
3 was carried out via a different oxidizing approach
either by oxidation of the tetrahydro-b-carboline deriv-
atives with sulfur in refluxing xylene, or with lead tetra-
acetate in glacial acetic acid, or with potassium
3
3–38
low oral bioavailability.
In this study, a series of novel b-carboline derivatives
were synthesized, and their cytotoxic activities were
O
O
O
OH
I
OCH3
OCH3
II
H
NH2
NH2
NH
N
N
H
N
H
H
CH3
2
chromatography
O
O
OCH3
OCH3
H
H
+
NH
NH
N
H
N
H
CH3
CH3
2
a
2b
Scheme 1. Synthetic route of methyl-1-methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylate (2a,b). Reagents and conditions: (I) thionyl chloride and
methanol; (II) sulfuric acid (cat) and aldehyde, rt.

7000
M. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 6998–7010
O
O
OH
OH
H
H
NH
+
NH
N
N
H
H
CH3
CH3
1
a
1b
chromatography
O
O
O
OH
OH
I
OCH3
H
II
H
NH2
NH
NH
N
H
N
N
H
H
CH3
CH3
1
2
chromatography
O
O
OCH3
OCH3
H
H
+
NH
NH
N
H
N
H
CH3
CH3
2a
2b
Scheme 2. Alternative synthetic route of methyl-1-methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylate (2a,b). Reagents: (I) sulfuric acid and
aldehyde; (II) thionyl chloride and methanol.
O
NHNH2
N
N
H
CH3
6
O
O
IV
O
O
OCH3
OCH3
OH
AA OBzl
H
I
II
III
NH
N
N
N
N
H
N
H
N
H
N
H
CH3
CH3
CH3
CH3
4
5a-m
3
2
O
O
V
O
NHCH CH NH
NH(CH ) NH
2
+
2
2
2
2
N
N
N
H
N
H
N
H
CH3
CH3
7
8
Scheme 3. Synthesis of b-carboline derivatives 5a–n, 6–8. Reagents and conditions: (I) potassium hypermanganate/DMF or sulfur flour/xylene or
lead tetraacetate/HOAc; (II) sodium hydroxide (aq); (III) DCC/DMAP, HCl–Gly-OBzl for 5a; HCl–Ala-OBzl for 5b; HCl–VaL-OBzl for 5c; HCl–
Phe-OBzl for 5d; HCl–Thr-OBzl for 5e; HCl–Tyr-OBzl for 5f; HCl-Leu-OBzl for 5g; HCl–Ile-OBzl for 5h; HCl–Pro-OBzl for 5i; HCl–Glu-OBzl for
2 2 2 2 2 2 2
5j; HCl–Asp-OBzl for 5k; HCl–Ser-OBzl for 5l; HCl–Lys(Dde)-OBzl for 5m; HCl–Arg-OBzl for 5n. (IV) NH NH H O; (V) NH CH CH NH . In
5a: AA = Gly; in 5b: AA = Ala; in 5c: AA = Val; in 5d: AA = Phe; in 5e: AA = Thr; in 5f: AA = Tyr; in 5g: AA = Leu; in 5h: AA = Ile; in 5i:
AA = Pro; in 5j: AA = Glu; in 5k: AA = Asp; in 5l: AA = Ser; in 5m: AA = Lys; in 5n: AA = Arg.
hypermanganate in DMF. Sulfur oxidation has the
highest (65%) yield. Subsequently, compound 3 was
hydrolyzed in the presence of sodium hydroxide to pro-
vide the key intermediate 4 (82% yield), 1-methyl-b-
carboline-3-carboxylic acid, which was then subjected
to the coupling reaction to generate a series of b-carbo-
line amino acid ester conjugates 5a–n (8–53% yield).
For the comparison with b-carboline Lys/Arg conjugate,
the diamine chains were introduced to the b-carboline
system. Refluxing of compound 3 with 85% hydrazine
hydrate in methanol generated the hydrazide 6 with
38% yield. Similarly, the amidation of compound 3
and ethylenediamine yielded compound 7 (71% yield)
and bis-b-carboline derivative 8 with 2% yield.

M. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 6998–7010
7001
2
derivatives
.2. In vitro cytotoxicity screening of b-carboline
lines (e.g., Ala and Val conjugates with IC50 values of 19
and 38 lM in HeLa cells). Similarly, when comparing
Ser to Thr conjugate, it was also noticed that an extra
carbon in the amino acid side chain caused a marked
difference in the individual activity profile (Table 1).
This observation implied the hydrophobic nature of
the side chains might influence the potency of the amino
acid ester conjugates. Both of the Leu and Ile conjugates
displayed comparably effective cytotoxic activity against
HeLa cell line (Leu: IC50 = 36 lM; Ile: IC50 = 27 lM).
Glu conjugate showed improved activity against all the
cell lines tested with IC50 values of 35, 46, and 50 lM.
The Phe and Tyr appeared to be particularly interesting
because these aromatic residues can intercalate with
The cytotoxic potential of all synthesized b-carboline
derivatives was evaluated against a panel of human
tumor cell lines using MTT (3-[4,5-dimethylthiazoL-2-yl]-
2,5-diphenyltetrazolium bromide) assay to determine
the drug concentration required to inhibit the growth
3
9–41
of human cancer cells by 50% (IC ) after 72 h.
5
The IC50 results are summarized in Table 1.
0
Our efforts to improve the cytotoxic activity of b-carbo-
line derivatives resulted in the compounds 5a–n and 6–8.
At the 3-position of b-carboline ring, we introduced the
different amino acids to yield a series of b-carboline ami-
no acid ester conjugates 5a–n. These amino acids pro-
vide a variety of functional groups, which including
the cationic residue (Lys, Arg), aromatic (Tyr, Trp,
Phe), anionic (Glu, Asp), and neutral aliphatic (Ser,
Thr, Gly, Ala, Leu, Val, Ile). Interestingly, as shown
in Table 1, compared with the parent b-carbolines (3:
IC50 = 121–167 lM; 4: IC50 = 140–189 lM), introduc-
tion of amino acids to the b-carboline core showed en-
hanced cytotoxic activity (except for Tyr-, Pro
conjugate). The incorporation of Gly showed moderate
improvement in cytotoxicity activities against HeLa,
MCF-7, and HepG-2 with IC50 values of 50, 48, and
3
1,42–44
DNA.
However, the respective contribution from
the Phe/Tyr residue to the cytotoxic activity was quite
different. The Phe conjugate, as expected, indeed dem-
onstrated better cytotoxic activity against HeLa,
MCF-7, and HeG2 cell lines, with IC50 values of 12,
14, and 25 lM, respectively. Whereas in the case of
Tyr conjugate, reduced cytotoxic activity regarding the
tested cell lines was observed with IC50 higher than
100 lM. In the case of the Pro conjugate, least improved
cytotoxic activity was observed with IC50 values of 85,
120, and 100 lM, respectively, against the cell lines
tested.
4
5 lM, respectively. While other neutral aliphatic amino
A potent cytotoxicity activity was observed with the
Lys/Arg conjugates. In the present study, regarding the
activity against individual cell lines, Lys and Arg conju-
gates are the most active compounds toward the tumor
cell lines tested. Lys and Arg conjugate showed the high-
est cytotoxic activity among all of the b-carboline amino
acid ester conjugates toward HeLa cell line at IC50
values of 4 and 1 lM, respectively. Clearly, introducing
Arg or Lys residue into the b-carboline dramatically
enhanced its cytotoxic activity. It appears that the basic
residues of Lys and Arg are crucial for DNA binding,
and the positively charged amino acids might provide
important structural contributions to the interaction
with DNA. We speculate that the side chain of Arg/
Lys was well suited to binding region on DNA because
of its flexibility, its potential to form ionic bonds with
the phosphate backbone and to donate multiple hydro-
gen bonds. Moreover, the guanidinium group of Arg
can potentially interact with aromatic ring structures
of DNA bases via its planar surface of shared p elec-
acid conjugates endowed with good cytotoxic activity
against HeLa cell lines. (Thr: IC50 = 27 lM; Ala:
IC50 = 19 lM; Leu: IC50 = 36 lM; Val: IC50 = 38 lM;
Ile: IC50 = 27 lM). Although Ala and Val conjugates
share a similar hydrophobic residue, they exhibited
some difference in cytotoxic activity against the same cell
Table 1. In vitro cytotoxic activities of b-carboline derivatives (IC50
lM)
,
a,c
b
b
b
Compound
HeLa
MCF-7
HepG2
3
4
121
140
50
19
38
12
27
108
36
27
85
35
51
68
4
167
180
48
30
54
14
23
153
45
39
120
46
70
34
2
135
189
45
49
44
25
53
146
63
57
100
50
49
53
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
6
7
8
a (Gly)
b (Ala)
c (Val)
d (Phe)
e (Thr)
f (Tyr)
g (Leu)
h (Ile)
3
1,42–44
trons.
In addition, the cationic amino acid side
chain would not preclude from functioning as DNA
3
1,42–44
i (Pro)
j (Glu)
k (Asp)
l (Ser)
minor groove directed component.
In this regard,
it is conceivable to envision that b-carboline Arg conju-
gate acts as a complex of DNA intercalator and minor
groove binder to initiate the amplification of DNA bind-
ing affinity and cytotoxic activity. The biological profile
of b-carboline Arg conjugate was significantly improved
by the incorporation of positively charged side chain of
Arg. This observation was consistent with the previous
suggestion that ‘Arg is a generic DNA binding amino
acid residue that creates or enhances DNA binding in
m (Lys)
n (Arg)
1
5
7
66
59
80
94
71
110
52
62
103
a
Cytotoxicity as IC50 for each cell line is the concentration of com-
pound, which reduced by 50% the optical density of treated cells with
respect to untreated cells using the MTT assay.
4
5–47
a variety of contexts.’
b
c
Cell lines include liver carcinoma (HepG-2), cervical carcinoma
(HeLa), and human breast cancer (MCF-7).
The data represent means ± SEM from at least three determinations.
To further investigate the effect of substitutions on the
cytotoxicity of b-carboline derivatives, we designed

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M. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 6998–7010
and synthesized compounds 6, 7, and 8. Regarding the
individual cell lines, compound 6 showed moderate
cytotoxicity with IC50 values of 66, 94, and 52 lM,
respectively, and compound 7 exhibited slightly better
activity than compound 6, with IC50 values of 59, 71,
and 62 lM. Furthermore, it was noticed the attachment
of second bulk hydrophobic b-carboline appeared not
beneficial to the cytotoxic activity. For example, com-
pared with compound 7, compound 8 has shown de-
creased cytotoxic activity with IC50 values of 80, 110,
and 103 lM. The decreased cytotoxicity might be due
to the steric effect.
Table 2. The apparent permeability coefficients (Papp) of 4 and 5b, d, e,
k, m, n
ꢀ
6
Compound
Papp · 10 cm/s
A ! B
B ! A
(A ! B)/ (B ! A)
4
7.95
10.25
11.53
11.19
8.33
7.80
6.34
6.21
6.32
8.11
6.10
6.00
1.02
1.62
1.86
1.77
1.03
1.97
2.01
5
5
5
5
b
d
e
k
5m
5n
12.03
12.11
The standard deviations were generally less than 10% (n = 4); A ! B:
from apical side to basolateral side; B ! A: from basolateral side to
apical side.
The in vitro cytotoxicity experiment indicated a subtle
change in the size, shape, and polarity of the substitu-
tion at position-3 of b-carboline derivatives can cause
a large change in its biological profile. This observation
may imply the spatial orientation and mobility of the
pendant group is important in DNA recognition and
The influence of efflux carriers on the permeability of
the different AA ester conjugates was also examined
by comparing the permeability ratio Pratio of absorptive
transport Papp (A ! B) to the secretory one Papp
4
8
binding.
4
9–54
(B ! A).
2
.3. In vitro membrane permeation study
According to the previous study, the compound with
,
ꢀ6
ꢀ6
Oral delivery is a noninvasive method which is a pre-
ferred route for drug administration. To elicit their
pharmacological and therapeutic effects, drug molecules
first have to enter the systemic blood circulation, which
requires the passage through the barrier membranes of
the gastrointestinal tract. The prediction of drug-mem-
brane permeability is important during the lead optimi-
zation stage of drug discovery. The experimental
difficulty, high cost, and low-throughput involved in
screening lead compounds in animals for oral drug
absorption have led to the development of several in
vitro prediction models. Caco-2 model has been widely
used for the prediction of oral absorption in
permeability coefficients Papp < 1 · 10 , 1-10 · 10
ꢀ6
and >10 · 10 cm/s is defined as poorly, moderately,
5
1
and well absorbed, respectively. From the permeability
coefficient (Papp) data, as observed in Table 2, the com-
pounds 5b, d, e, m, n, that is, Ala-, Phe-, Thr-, Lys-, Arg-
conjugate was identified as well-absorbed compound. By
comparison of the apparent permeability coefficients
P
app of the amino acid conjugates 5b, d, e, m, n, with
the parent compound 4, the transepithelial transport
of the amino acid conjugates across the Caco-2 cell
monolayer in the absorptive (A ! B) direction, and
their respective P
coefficients were higher than in
app
the secretory (B ! A) direction, which indicated the
amino acid moiety in the b-carboline derivatives was
favorable for the active transport across the biomem-
branes. In particular, the Papp (A ! B) values of
Lys- (5m) and Arg conjugate (5n) are higher than their
corresponding Papp (B ! A) values, and the permeabil-
4
9–51
humans.
The membranes of Caco-2 cells have useful
properties for correction with in vivo data and it is wide-
ly used for investigating transport, efflux, paracellular
and transcellular diffusion, and metabolic activity dur-
5
2,53
ing absorption.
ity ratio of Papp (A ! B)/P
(B ! A) is close to
app
In our study, Caco-2 cell line monolayer was used as
intestinal absorption model for screening b-carboline
amino acid ester conjugates, and thus for evaluating
the amino acid conjugation approach for enhanced drug
absorption. Drug permeability is difficult to measure,
thus it is simpler to estimate by the apparent permeabil-
2. These asymmetric permeation profiles implied AA
conjugates might not be the substrates of the apically
localized efflux P-gp carrier, which may limit their oral
bioavailability by transporting the absorbed drug back
into the intestinal lumen. However, additional experi-
ments are required for evaluating the beneficial effect
of amino acid conjugates on the transport.
5
1
ity coefficient (Papp).
Papp (A ! B) is the permeability from the apical to the
basolateral side (intestine to blood), and Papp (B ! A)
is the permeability from the basolateral to the apical side
2.4. Molecular modeling
Molecule modeling studies indicated that b-carboline
amino acid ester conjugates might interact with DNA
by intercalating, and consequently inhibit DNA synthe-
sis. b-carboline Arg conjugate 5n bearing guanidium
group at the side chain had generally lower energy than
the other conjugates. In particular, it was observed that
b-carboline Arg conjugate slightly induced a conforma-
tional change in DNA suggesting that one key arginine
residue or guanidine might contribute to trigger the min-
or conformational change in DNA. b-carboline Arg
conjugate docking in DNA as an example is shown in
5
1,54
(
blood to intestine).
gates across Caco-2 cell monolayers were valuated in
The various amino acid conju-
triplicate in the apical to basolateral (A ! B) or basolat-
4
9–51
eral to apical directions (B ! A).
It was initiated by
adding the test solution to the apical or basolateral side
of the monolayer. Investigation of the transport across
the polarized Caco-2 cell monolayer in the basolateral
to apical direction constitutes a means of evaluating
the influence of P-gp and related efflux carriers which
5
1–54
are located on the apical side of the monolayer.

M. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 6998–7010
7003
Figure 1. The modeling study of Arg conjugate indicat-
ed that the planar b-carboline aromatic ring system had
been inserted into DNA such that the guanidine-termi-
nated side chain was forced to lie in the minor groove
of DNA. It seems that the relative positioning of side
chains in the b-carboline amino acid ester conjugates
may be an important factor in the DNA-drug
recognition.
From the molecular modeling analysis, we conclude that
the size and shape of the side chain of these conjugates
are crucial for high affinity and correct binding mode.
However, other interaction factors also contribute to
their distinct biological activities.
3. Conclusion
From the interaction energy data as shown in Table 3, it
was noticed that, b-carboline Arg conjugate exhibited
the lowest binding energy (5n: ꢀ40.9342 kcal/mol),
which indicated that b-carboline Arg conjugate could
form stable complex with DNA. It was noticed that
the rigid proline ring seemed to induce the steric hin-
drance and therefore resulting in the relative higher
binding energy in Pro conjugate (5i: ꢀ5.7609 kcal/
mol). In addition, it was clearly seen that, Tyr and Phe
conjugate exhibited similar binding energy (5f:
The in vitro cytotoxic activities of b-carboline
derivatives demonstrated b-carboline nucleus was an
important unit for the design and synthesis of new
anti-tumor drugs; the cytotoxicity of b-carboline
derivatives depended upon the presence and the nature
of the substitutions, which were introduced into the b-
carboline ring. The cytotoxic potencies of b-carboline
derivatives were enhanced by the introduction of amino
acids into 3-position of b-carboline ring. A cationic Lys/
Arg residue was highly favorable for the enhanced
cytotoxic activity compared to the uncharged amino
acids. A minor structural alternation in the single amino
acid chain of b-carboline amino acid ester conjugates
can lead to marked multiple changes in membrane
permeability, intercalating capability to the cytotoxicity,
which implied that the improved biological profile might
result from a synergic effect.
ꢀ
32.4253 kcal/mol; 5d: ꢀ30.1275 kcal/mol). Likewise,
it was observed in the case of Ser and Thr conjugate
with similar binding energy (5l: ꢀ20.8982 kcal/mol; 5e:
ꢀ
19.0324 kcal/mol). The reason for the differences in
their biological activities might be explained by the dif-
ferences in bioavailability such as drug uptake and dif-
ferent rate of metabolism.
Figure 1. Model illustrating b-carboline-Arg conjugate (compound 5n) docking in DNA.
Table 3. Computer simulation of the minimization binding energy b-carbolines amino acid conjugates and DNA
Compound
Phe (5d)
Thr (5e)
Tyr (5f)
Pro (5i)
ꢀ5.7609
Ser (5l)
Lys (5m)
ꢀ38.0016
Arg (5n)
E (kcal/mol)
ꢀ30.1275
ꢀ19.0324
ꢀ32.4253
ꢀ20.89822
ꢀ40.9342

7004
M. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 6998–7010
Introducing the amino acid moiety onto b-carboline
yielded a series of b-carboline amino acid ester conju-
gates representing one of the positive approaches to
optimize the lead compound. Clearly, further in vitro
and in vivo biological evaluation of b-carboline amino
acid conjugates as potential antitumor agents is needed
by application of mechanism-based bioassay systems,
such as the inhibition of human topoisomerase I and
II and molecular pathways that involved in cytotoxicity.
as a colorless powder. The filtrate was concentrated to
give 0.12 g (5%) of (1R,3S)-1-methyl-1,2,3,4-tetrahy-
dro-b-carboline-3-carboxylic acid as a colorless powder.
+
Compound 1a: Mp 290–292 ꢁC; ESI/MS: 231 [M+H] ;
IR (KBr): 3100–2400, 2960, 2900, 1700, 1620, 1590,
ꢀ
1 1
1500, 1450, 1379, 1070, 900 cm ; H NMR (DMSO-
d6): d = 11.94 (s, 1H), 10.99 (s, 1H), 9.18 (s, 1H), 7.44
(d, J = 7.5 Hz, 1H), 7.33 (t, J = 8.0 Hz, 1H), 7.08 (t,
J = 8.0 Hz, 1H), 6.99 (t, J = 7.5 Hz, 1H), 4.22 (q,
J = 4.8 Hz, 1H), 3.69 (dd, J = 10.5 Hz, J = 5.0 Hz,
1H), 3.14 (dd, J = 10.5 Hz, J = 2.4 Hz, 1H), 2.83 (ddd,
J = 10.5 Hz, J = 5.0 Hz, J = 2.4 Hz, 1H), 1.38 (d,
J = 5.0 Hz, 3H). Anal. Calcd for C H N O : C,
4. Experimental
4
.1. General
1
3
14
2
2
67.81; H, 6.13; N, 12.17. Found: C, 67.65; H, 6.32; N,
11.99.
The protected amino acids with L-configuration, used in
this work, were purchased from Sigma Chemical. H
1
1
3
+
NMR (500 Hz) and C NMR (75 Hz) spectra were
acquired on a Bruker AC 300 spectrometer in CDCl3
with TMS as internal standard, or in DMSO-d and
Compound 1b: Mp 240–242 ꢁC; ESI/MS: 231 [M+H] ;
IR (KBr): 3100–2400, 2960, 2900, 1700, 1620, 1590,
ꢀ
1 1
1500, 1450, 1379, 1070, 900 cm ; H NMR (DMSO-d ):
6
6
chemical shifts were expressed in ppm (d). All of the
coupling reactions were carried out under anhydrous
conditions. Chromatography was performed on Qing-
dao silica gel H. The purities of the intermediates and
the products were confirmed by TLC (Merck silica gel
plastes of type 60 F254, 0.25 mm layer thickness) and
HPLC (Waters, C18 column 4.6 · 150 mm). The amino
acid analysis was determined with a Hitachi 835-50
instrument. FAB-MS was determined by VG-ZAB-MS
high resolution GC/MS/DS and HP ES-5989x. Optical
rotations were determined with a Schmidt + Haensch
Polartromic D instrument. The statistical analysis of
all the biological data was carried out by use of ANOVA
test, p < 0.05 is considered significant.
d = 11.89 (s, 1H), 10.99 (s, 1H), 9.18 (s, 1H), 7.41 (d,
J = 7.6 Hz, 1H), 7.30 (t, J = 8.1 Hz, 1H), 7.01 (t,
J = 8.1 Hz, 1H), 6.97 (t, J = 7.6 Hz, 1H), 4.40 (q,
J = 4.9 Hz, 1H), 3.67 (dd, J = 10.3 Hz, J = 5.1 Hz,
1H), 3.16 (dd, J = 10.3 Hz, J = 2.7 Hz, 1H), 2.53 (ddd,
J = 10.3 Hz, J = 5.1 Hz, J = 2.6 Hz, 1H), 1.01 (d,
J = 5.1 Hz, 3H). Anal. Calcd for C H N O : C,
13
14
2
2
67.81; H, 6.13; N, 12.17. Found: C, 67.70; H, 6.00; N,
12.01.
4.4. Methyl (1S,3S)- and (1R,3S)-1-methyl-1,2,3,4-tetra-
hydro-b-carboline-3-carboxylate (2a,b)
(a) At 0 ꢁC, to the solution of methanol (10 ml), 1 ml of
thionyl chloride was added dropwise. The mixture was
stirred at rt for 15 min, and then 2.0 g (8.7 mmol) of
1-methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid
was added in portion. The reaction mixture was stirred
4.2. General procedure for preparation of amino acid
benzylester hydrochloride
To the solution of 10.0 g polyphosphoric acid and 50 ml
of benzyl alcohol, 0.02 mol amino acid was added. The
reaction mixture was stirred at 92 ꢁC for 8 h and then
mixed with the solution of 20 ml of concentrated sulfuric
acid in 200 ml of water. The mixture was treated with
at rt for 16 h until TLC (CHCl /MeOH, 15:1) indicated
3
the complete disappearance of 1-methyl-1,2,3,4-tetrahy-
dro-b-carboline-3-carboxylic acid. The reaction mixture
was neutralized with aqueous solution of sodium bicar-
bonate (10%) to pH 7.0. The formed precipitate was col-
lected by filtration. After separation with silica gel
chromatography (chloroform/acetone, 16:1), 1.88 g
(88.5%) of methyl (1S,3S)-1-methyl-1,2,3,4-tetrahydro-
b-carboline-3-carboxylate and 125 mg (5.5%) of methyl
(1R,3S)-1-methyl-1,2,3,4-tetrahydro-b-carboline-3-car-
boxylate were obtained as a pale yellow powder.
2
0 ml of ether, and the aqueous phase was separated
and then neutralized with aqueous solution of sodium
carbonate adjusted to pH 10. The solution was extracted
with ether (3· 70 ml) and the ether phase was dried with
anhydride magnesium sulfate. After filtration, the fil-
trate was bubbled with hydrogen chloride to precipitate
the hydrochloride of amino acid benzyl ester in 23–72%
yield as colorless powder.
+
Compound 2a: Mp 75–76 ꢁC; ESI/MS: 245 [M+H] ; IR
(KBr): 3400–3200, 2960, 2900, 2810, 1740, 1620, 1590,
1500, 1450, 1382, 1066, 897 cm ; H NMR (CDCl ):
ꢀ1 1
4.3. (1S,3S)- and (1R,3S)-1-methyl-1,2,3,4-tetrahydro-b-
carboline-3-carboxylic acid (1a, b)
3
d = 10.04 (s, 1H), 7.42 (t, J = 6.4 Hz, 1H), 7.20 (t,
J = 8.6 Hz, 1H), 7.11 (d, J = 7.5 Hz, 1H), 7.04 (d,
J = 6.6 Hz, 1H), 4.28 (q, J = 6.6 Hz, 1H), 3.78 (s, 3H),
3.77 (m, J = 6.9 Hz, 1H), 3.08 (d, J = 11.2 Hz, 1H), 2.81
(t, J = 11.2 Hz, 1H), 2.25 (s, 1H), 1.42 (d, J = 6.0 Hz,
3H). Anal. Calcd for C H N O : C, 68.83; H, 6.60; N
To the mixture of 2.0 g (9.9 mmol) of L-tryptophane,
.2 ml H SO (1 mol/L), and 80 ml of water, 2 ml of
2 4
0
aldehyde (40%) was added. The reaction mixture was
stirred at room temperature for 6 h and adjusted to
pH 6–7 with concentrated ammonia liquor. The mixture
was kept at 0 ꢁC for 12 h and the formed precipitate was
collected by filtration to give 1.8 g (80%) of (1S,3S)-1-
methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylic acid
1
4
16
2
2
11.47. Found: C, 68.67; H, 6.80; N, 11.64.
+
Compound 2b: Mp 187–188 ꢁC; ESI/MS: 245 [M+H] ;
IR (KBr): 3000, 2980, 2960, 2825, 1732, 1610, 1590,

M. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 6998–7010
7005
ꢀ
1
596, 1455, 1380, 1064, 899 cm ; H NMR (CDCl ):
1
1
J = 7.5 Hz, 1H), 6.97 (t, J = 6.4 Hz, 1H), 3.74 (s, 3H),
2.03 (s, 3H).
3
d = 10.08 (s, 1H), 7.40 (t, J = 7.1 Hz, 1H), 7.28 (d,
J = 8.4 Hz, 1H), 7.08 (t, J = 7.7 Hz, 1H), 7.00 (t,
J = 6.5 Hz, 1H), 4.37 (q, J = 6.5 Hz, 1H), 3.98 (t,
J = 4.9 Hz, 1H), 3.74 (s, 3H), 3.08 (q, J = 4.9 Hz, 1H),
2.95 (q, J = 6.6 Hz, 1H), 2.75 (s, 1H), 1.47 (d,
J = 6.4 Hz, 3H). Anal. Calcd for C H N O : C,
(c) At 0 ꢁC, with stirring to the solution of 5.0 g
(20.0 mmol) of methyl-1-methyl-1,2,3,4-tetrahydro-b-
carboline-3-carboxylate in 10 ml of glacial acetic acid,
45.0 g (0.1 mol) of lead tetraacetate was added. After
stirring at room temperature for 15 min to the reaction
mixture, 10.0 g (0.1 mol) of oxalic acid was added. The
reaction mixture was stirred at room temperature for
1 h and TLC (chloroform/methanol, 15:1) indicated
the complete disappearance of methyl-1-methyl-1,2,3,4-
tetrahydro-b-carboline-3-carboxylate. The formed pre-
cipitate was collected by filtration and then suspended
in 150 ml of mixed solvent of water and chloroform
(1:2). The suspension was neutralized with sodium bicar-
bonate and filtered. The filtered residue was washed with
20 ml of chloroform and the filtrate was extracted with
chloroform (310 ml). The combined chloroform was
dried with anhydrous sodium sulfate and filtered. The
filtrate was evaporated to give 2.5 g (50%) of the title
compound which was obtained as a yellow powder.
1
4
16
2
2
6
1
8.83; H, 6.60; N, 11.47. Found: C, 68.99; H, 6.79; N,
1.31.
(b) To the mixture of 2.2 g (9.9 mmol) of L-tryptophane
methyl ester and 0.1 ml H SO in 80 ml of water, 2 ml of
2
4
aldehyde (40%) was added. The reaction mixture was
stirred at rt for 6 h and adjusted to pH 6–7 with concen-
trated ammonia liquor. The mixture was kept at 0 ꢁC for
1
2 h and the formed precipitate was collected by filtra-
tion to give 1.18 g (47%) of methyl 1-methyl-1,2,3,4-tet-
rahydro-b-carboline-3-carboxylate. After separation
with silica gel chromatography (chloroform/acetone,
6:1), 787 mg (31%) of methyl (1S,3S)-1-methyl-
,2,3,4-tetrahydro-b-carboline-3-carboxylate and 394
1
1
(16%) of methyl (1R,3S)-1-methyl-1,2,3,4-tetrahydro-b-
carboline-3-carboxylate were obtained as a colorless
+
Mp 242–243 ꢁC; ESI/MS: 241 [M+H] ; IR (KBr):
3310, 2954, 2922, 2901, 2811, 1742, 1600, 1581, 1566,
powder.
ꢀ
1
1
1450, 1380, 1066, 900 cm
;
H NMR (DMSO-d6):
4
.5. Methyl-1-methyl-b-carboline-3-carboxylate (3)
d = 9.98 (s, 1H), 7.41 (d, J = 5.6 Hz, 1H), 7.35 (d,
J = 7.2 Hz, 1H), 7.15 (d, J = 8.2 Hz, 1H), 7.11 (t,
J = 7.5 Hz, 1H), 6.97 (t, J = 6.4 Hz, 1H), 3.74 (s, 3H),
2.03 (s, 3H).
(a) At 0 ꢁC with stirring to the solution of 5.0 g
20.0 mmol) of (1S,3S)-methyl-1-methyl-1,2,3,4-tetrahy-
(
dro-b-carboline-3-carboxylate in 50 ml DMF, 4.5 g
28.0 mmol) of potassium hypermanganate was added.
(
4.6. 1-Methyl-b-carboline-3-carboxylic acid (4)
The reaction mixture was stirred at 0 ꢁC for 1 h and then
at room temperature for 14 h, and TLC (chloroform/
methanol, 15:1) indicated the complete disappearance
of methyl-1-methyl-1,2,3,4-tetrahydro-b-carboline-3-
carboxylate. After evaporation, the residue was dis-
The solution of 5.0 g (20.8 mmol) of methyl 1-methyl-b-
carboline-3-carboxylate in 120 ml of aqueous solution of
sodium hydroxide (2.0 mmol/L) was stirred at 60 ꢁC for
4 h and TLC (chloroform/methanol, 15:1) indicated the
complete disappearance of methyl-1-methyl-b-carbo-
line-3-carboxylate. The reaction mixture was neutralized
with hydrochloric acid to pH 7.0. The formed precipi-
tate was collected by filtration to give 3.86 g (82%) of
the title compound as a yellow powder. Mp 292–
solved in 10 ml of methanol, filtered, and concentrated,
3
.2 g (65%) of the title compound was obtained as yel-
+
low powder. Mp 243–244 ꢁC; ESI/MS: 241 [M+H] ;
IR (KBr): 3310, 2954, 2922, 2901, 2811, 1742, 1600,
ꢀ1
1
1
581, 1566, 1450, 1380, 1066, 900 cm
;
H NMR
+
(
CDCl ): d = 9.98 (s, 1H), 7.41 (d, J = 5.6 Hz, 1H),
293 ꢁC; ESI/MS: 227 [M+H] ; IR (KBr): 3230–2252,
2924, 2910, 2900, 1700, 1610, 1585, 1562, 1440, 1380,
3
7
(
.35 (d, J = 7.2 Hz, 1H), 7.15 (d, J = 8.2 Hz, 1H), 7.11
t, J = 7.5 Hz, 1H), 6.97 (t, J = 6.4 Hz, 1H), 3.74 (s,
ꢀ
1 1
1066, 900 cm ; H NMR (CDCl ): d = 11.06 (s, 1H),
3
3
6
1
H), 2.03 (s, 3H). Anal. Calcd for C H N O : C,
2
9.99; H, 5.03; N, 11.66. Found: C, 69.80; H, 5.20; N,
1.82.
9.88 (s, 1H), 7.42 (d, J = 5.8 Hz, 1H), 7.39 (d,
J = 7.1 Hz, 1H), 7.10 (d, J = 8.0 Hz, 1H), 7.10 (t,
J = 7.6 Hz, 1H), 6.99 (t, J = 6.6 Hz, 1H), 2.10 (s, 3H).
Anal. Calcd for C H N O : C, 69.02; H, 4.46; N,
1
4
12
2
1
3
10
2
2
(
b) The suspension of 5.0 g (20.0 mmol) of methyl-1-
methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylate,
.4 g (20. mmol) sulfur flour, and 20 ml of anhydrous
dimethyl benzene was refluxed for 96 h until TLC
chloroform/methanol, 15:1) indicated the complete
12.38. Found: C, 69.20; H, 4.60; N, 12.22.
1
4.7. N-(1-Methyl-b-carboline-3-carbonyl)glycine benzyl
ester (5a)
(
disappearance of methyl 1-methyl-1,2,3,4-tetrahydro-
b-carboline-3-carboxylate. On evaporation the residue
was dissolved in 10 ml of methanol. After filtration
and evaporation under reduced pressure, 3.2 g (65%)
of the title compound was obtained as a yellow powder.
At 0 ꢁC, to the solution of 364 mg (2.0 mmol) HCl–Gly-
OBzl, 0.5 ml (4.5 mmol) of N-methylmorpholine,
454 mg (2.2 mmol) DCC, DMAP 23 mg (0.2 mmol),
5 ml of anhydrous THF, and 4 ml of anhydrous
DMF, 452 mg (2.0 mmol) of 1-methyl-b-carboline-3-
carboxylic acid was added. The reaction mixture was
stirred at 0 ꢁC for 1 h, at rt for another 16 h until TLC
(chloroform/methanol, 15:1) indicated the complete dis-
appearance of 1-methyl-b-carboline-3-carboxylic acid.
The reaction mixture was filtered, concentrated, and
+
Mp 242–243 ꢁC; ESI/MS: 241 [M+H] ; IR (KBr): 3310,
2
1
954, 2922, 2901, 2811, 1742, 1600, 1581, 1566, 1450,
380, 1066, 900 cm
ꢀ1
1
;
H NMR (500, DMSO-d6):
d = 9.98 (s, 1H), 7.41 (d, J = 5.6 Hz, 1H), 7.35 (d,
J = 7.2 Hz, 1H), 7.15 (d, J = 8.2 Hz, 1H), 7.11 (t,

7006
M. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 6998–7010
the residue was dissolved in 40 ml of ethyl acetate and
washed successively with 5% sodium bicarbonate, 5%
citric acid, and saturated sodium chloride, and the
organic phase was separated and dried over anhydrous
sodium sulfate. After filtration and evaporation under
reduced pressure, 280 mg (37%) of the title compound
was obtained as a colorless powder. Mp 156–158 ꢁC;
8.70 (s, 1H), 8.24 (s, 1H), 7.70 (d, J = 7.2 Hz, 1H),
7.57 (d, J = 7.5 Hz, 1H), 7.16 (t, J = 7.2 Hz, 1H), 7.06
(t, J = 7.6 Hz, 1H), 7.32 (t, J = 7.6 Hz, 1H), 7.30 (t,
J = 7.8 Hz, 1H), 7.29 (t, J = 7.5 Hz, 2H), 7.28 (t,
J = 7.6 Hz, 2H), 7.27 (d, J = 7.6 Hz, 2H), 7.26 (d,
J = 7.5 Hz, 2H), 5.26 (s, 2H), 5.14 (dd, J = 4.8 Hz,
J = 3.5 Hz, 1H), 3.36 (d, J = 4.8 Hz, 2H), 2.72 (s, 3H).
+
20
ESI/MS: 374 [M+H] . IR (KBr) 3341, 3028, 3009,
½aꢁ 3.0 (c 0.5, EtOH/DMSO, 4:1). Anal. Calcd for
D
C H N O : C, 75.14; H, 5.44; N, 9.07. Found: C,
75.01; H, 5.26; N, 9.20.
ꢀ
1 1
1
751, 1690, 1601, 1582, 1501, 1460, 762, 701 cm . H
2
9
25
3
3
NMR (CDCl ) d = 12.01 (s, 1H), 8.70 (s, 1H), 8.33 (s,
3
1
7
H), 7.72 (d, J = 7.2 Hz, 1H), 7.48 (d, J = 7.5 Hz, 1H),
.32 (d, J = 8.4 Hz, 2H), 7.29 (t, J = 7.2 Hz, 2H), 7.20
4.11. N-(1-Methyl-b-carboline-3-carbonyl)-L-threonine
benzyl ester (5e)
(
t, J = 7.8 Hz, 1H), 7.12 (t, J = 7.2 Hz, 1H), 7.07 (t,
J = 7.5 Hz, 1H), 5.28 (s, 2H), 4.01 (d, J = 5.4 Hz, 2H),
.82 (s, 3H). Anal. Calcd for C H N O : C, 70.76;
2
Using the similar procedure as that of 5a, starting from
454 mg (2.0 mmol) of HCl–L-Thr-OBzl, 126 mg (15%)
of the title compound was obtained as a colorless pow-
2
2
19
3
3
H, 5.13; N, 11.25. Found: C, 70.68; H, 5.22; N, 11.40.
+
4.8. N-(1-Methyl-b-carboline-3-carbonyl)-L-alanine
benzyl ester (5b)
der. Mp 178–179 ꢁC; ESI/MS: 418 [M+H] ; IR (KBr)
3339, 3028, 3009, 1754, 1690, 1603, 1581, 1502, 1465,
ꢀ
1
1
7
61, 702 cm . H NMR (CDCl ) d = 12.02 (s, 1H),
3
Using the similar procedure as that of 5a, starting from
392 mg (2.0 mmol) of HCl–L-Ala-OBzl, 154 mg (20%) of
the title compound was obtained as a colorless powder.
Mp 158–160 ꢁC; ESI/MS: 388 [M+H] . IR(KBr)3343,
8.79 (s, 1H), 8.22 (s, 1H), 7.73 (d, J = 7.8 Hz, 1H),
7.55 (t, J = 7.5 Hz, 1H), 7.45 (d, J = 7.2 Hz, 1H), 7.35
(t, J = 7.8 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 7.30 (t,
J = 7.6 Hz, 2H), 7.27 (d, J = 7.5 Hz, 2H), 5.29 (s, 2H),
+
3
7
1
1
7
032, 3008, 1754, 1690, 1605, 1582, 1505, 1464, 762,
4.63 (m, J = 5.2 Hz, 1H), 4.52 (d, J = 5.2 Hz, 1H), 2.85
ꢀ
1
1
20
D
(c 0.5, EtOH/DMSO, 4:1). Anal. Calcd for
01 cm . H NMR (CDCl ) d = 12.02 (s, 1H), 8.70 (s,
(s, 3H), 2.22 (s, 1H), 1.66 (d, J = 5.6 Hz, 3H). ½aꢁ 4.0
3
H), 8.43 (s, 1H), 8.04 (s, 1H), 7.59 (d, J = 7.5 Hz,
H), 7.46 (d, J = 8.4 Hz, 1H), 7.41 (d, J = 8.2 Hz, 2H),
.27 (t, J = 7.3 Hz, 2H), 7.22 (t, J = 7.7 Hz, 1H), 7.05
C H N O : C, 69.05; H, 5.55; N, 10.07. Found: C,
2
4
23
3
4
68.88; H, 5.27; N, 10.21.
(
1
(
t, J = 7.4 Hz, 1H), 5.29 (s, 2H), 4.91 (m, J = 6.4 Hz,
20
D
c 0.5, EtOH/DMSO, 4:1), Anal. Calcd for C H N O :
H), 2.85 (s, 3H), 1.66 (d, J = 6.6 Hz, 3H). ½aꢁ 2.0
4.12. N-(1-Methyl-b-carboline-3-carbonyl)-L-tyrosine
benzyl ester (5f)
2
3
21
3 3
C, 71.30; H, 5.46; N, 10.85. Found: C, 71.49; H, 5.32;
N, 10.66.
Using the similar procedure as that of 5a, starting from
578 mg (2.0 mmol) of HCl–L-Tyr-OBzl, 197 mg (20%) of
the title compound was obtained as a colorless powder.
4.9. N-(1-Methyl-b-carboline-3-carbonyl)-L-valine benzyl
ester (5c)
+
Mp 136–137 ꢁC; ESI/MS: 480 [M+H] ; IR (KBr) 3342,
3
701 cm
031, 3012, 1754, 1693, 1605, 1582, 1501, 1460, 762,
.
ꢀ
1
1
Using the similar procedure as that of 5a, starting from
H NMR (DMSO-d ) d = 12.02 (s, 1H),
6
4
50 mg (2.0 mmol) of HCl–L-VaL-OBzl, 183 mg (22%)
of the title compound was obtained as a colorless pow-
8.79 (s, 1H), 8.22 (s, 1H), 7.63 (d, J = 7.8,1H), 7.55 (d,
J = 7.8 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 7.34 (t,
J = 7.5 Hz, 2H), 7.32 (d, J = 7.8 Hz, 2H), 7.10 (t,
J = 7.6 Hz, 1H), 7.07 (d, J = 7.5 Hz, 1H), 6.99 (d,
J = 6.9 Hz, 2H), 6.61 (d, J = 6.8 Hz, 2H), 5.26 (s, 2H),
5.06 (s, 1H), 4.51 (t, J = 4.5 Hz, 1H), 3.14 (d,
+
der. Mp 162–164 ꢁC; ESI/MS: 416 [M+H] ; IR (KBr)
3
7
8
7
338, 3025, 3009, 1751, 1690, 1602, 1581, 1504, 1460,
ꢀ
1
1
61, 698 cm . H NMR (CDCl ) d = 11.68 (s, 1H),
3
.38 (s, 1H), 8.04 (s, 1H), 7.57 (d, J = 7.5 Hz, 1H),
.56 (d, J = 7.2 Hz, 1H), 7.31 (t, J = 7.8 Hz, 2H), 7.29
20
J = 4.5 Hz, 2H), 2.82 (s, 3H). ½aꢁ ꢀ25.0 (c 0.5, EtOH/
D
DMSO, 4:1). Anal. Calcd for C H N O : C, 72.64;
H, 5.25; N, 8.76. Found: C, 72.80; H, 5.44; N, 8.59.
(
d, J = 7.6 Hz, 2H), 7.26 (d, J = 7.5 Hz, 1H), 7.15 (t,
J = 7.5 Hz, 1H), 7.05 (t, J = 7.5 Hz, 1H), 5.29 (s, 2H),
.91 (d, J = 7.2 Hz, 1H), 2.85 (s, 3H), 2.22 (m,
2
9
25
3
4
4
20
J = 7.2 Hz, 1H), 1.66 (d, J = 6.6 Hz, 6H). ½aꢁ 5.0
4.13. N-(1-Methyl-b-carboline-3-carbonyl)-L-leucine
benzyl ester (5g)
D
c 0.5, EtOH/DMSO, 4:1). Anal. Calcd for C H N O :
(
2
5
25
3 3
C, 72.27; H, 6.06; N, 10.11. Found: C, 72.46; H, 6.22;
N, 10.30.
Using the similar procedure as that of 5a, starting from
478 mg (2.0 mmol) of HCl–L-Leu-OBzl, 256 mg (30%)
of the title compound was obtained as a colorless pow-
4.10. N-(1-Methyl-b-carboline-3-carbonyl)-L-phenylala-
nine benzyl ester (5d)
+
der. Mp 183–185 ꢁC; ESI/MS: 430 [M+H] ; IR(KBr)
3
762, 701 cm
338, 3027, 3012, 1749, 1689, 1601, 1582, 1502, 1461,
.
ꢀ
1
1
Using the similar procedure as that of 5a, starting from
546 mg (2.0 mmol) of HCl–L-Phe-OBzl, 490 mg (53%)
of the title compound was obtained as a colorless powder.
H NMR (DMSO-d ) d = 12.00 (s,
6
1H), 8.67 (s, 1H), 8.22 (s, 1H), 7.67 (d, J = 8.7 Hz,
1H), 7.54 (d, J = 7.8 Hz, 1H), 7.34 (d, J = 7.8 Hz, 2H),
7.32 (t, J = 7.5 Hz, 2H), 7.30 (t, J = 7.8 Hz, 1H), 7.13
(t, J = 7.6 Hz, 1H), 7.01 (t, J = 7.5 Hz, 1H), 5.26 (s,
2H), 4.60 (t, J = 5.1 Hz, 1H), 2.84 (s, 3H), 1.99 (m,
+
Mp 138–140 ꢁC; ESI/MS: 464 [M+H] ; IR (KBr) 3342,
3
7
032, 3011, 1748, 1690, 1601, 1581, 1502, 1465,
ꢀ
1
1
61, 702 cm . H NMR (CDCl ) d = 12.02 (s, 1H),
3

M. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 6998–7010
7007
J = 5.1 Hz, 2H), 1.20(m, J = 5.0 Hz, 1H), 0.96 (d,
4.17. N-(1-Methyl-b-carboline-3-carbonyl)-L-aspartic
acid benzyl ester (5k)
20
J = 6.0 Hz, 6H). ½aꢁ 4.0 (c 0.5, EtOH/DMSO, 4:1).
D
Anal. Calcd for C H N O : C, 72.71; H, 6.34; N,
26
27
3
3
9
.78. Found: C, 72.90; H, 6.49; N, 9.59.
Using the similar procedure as that of 5a, starting
from 660 mg (2.0 mmol) of HCl–L-Asp-OBzl, 100 mg
(10%) of the title compound was obtained as a color-
4
benzyl ester (5h)
.14. N-(1-Methyl-b-carboline-3-carbonyl)-L-isoleucine
+
less powder. Mp 202–204 ꢁC; ESI/MS: 522 [M+H] ;
IR (KBr) 3342, 3027, 3009, 1754, 1690, 1601, 1582,
ꢀ
1
1
Using the similar procedure as that of 5a, starting from
1501, 1463, 760, 704 cm
.
H NMR (DMSO-d6)
4
78 mg (2.0 mmol) of HCl–L-Ileu-OBzl, 346 mg (40%)
of the title compound was obtained as a colorless pow-
d = 12.01 (s, 1H), 8.70 (s, 1H), 8.30 (s, 1H), 7.69 (d,
J = 7.8 Hz, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.34 (t,
J = 7.3 Hz, 2H), 7.32 (t, J = 7.3 Hz, 2H), 7.30 (t,
J = 7.8 Hz, 2H), 7.28 (t, J = 7.8 Hz, 2H), 7.26 (t,
J = 7.8 Hz, 1H), 7.24 (t, J = 7.8 Hz, 1H), 7.18 (t,
J = 7.6 Hz, 1H), 7.01 (t, J = 7.6 Hz, 1H), 5.28 (s,
2H), 5.25 (s, 2H), 4.87 (m, J = 6.2 Hz, 1H), 3.20 (m,
+
der. Mp 182–183 ꢁC; ESI/MS: 430 [M+H] ; IR (KBr)
3
1
d = 12.02 (s, 1H), 8.72 (s, 1H), 8.20 (s, 1H), 7.55 (d,
J = 8.7 Hz, 1H), 7.34 (d, J = 7.8 Hz, 1H), 7.30 (d,
J = 8.1 Hz, 1H), 7.28 (t, J = 7.5 Hz, 2H), 7.24 (t,
J = 7.8 Hz, 1H), 7.12 (t, J = 7.5 Hz, 1H), 7.01 (t,
J = 7.6 Hz, 1H), 5.26 (s, 2H), 4.51 (d, J = 5.1 Hz, 1H),
341, 3032, 3011, 1751, 1690, 1602, 1581, 1502, 1461,
H NMR (DMSO-d6)
ꢀ
1
.
1
380, 1371, 761, 701 cm
20
J = 6.2 Hz, 2H), 2.81 (s, 3H). ½aꢁ
4.0 (c = 0.5,
EtOH/DMSO, 4:1). Anal. Calcd for C H N O : C,
D
3
1
27
3
5
71.39; H, 5.22; N, 8.06. Found: C, 71.22; H, 5.03;
N, 8.23.
2
J = 5.1 Hz, 2H), 0.97 (d, J = 6.9 Hz, 3H), 0.93 (m,
.85 (s, 3H), 2.83 (m, J = 5.1 Hz, 1H), 1.29 (m,
20
J = 6.9 Hz, 3H). ½aꢁ 3.0 (c 0.5, EtOH/DMSO, 4:1).
4.18. N-(1-Methyl-b-carboline-3-carbonyl)-L-serine ben-
zyl ester(5l)
D
Anal. Calcd for C H N O : C, 72.71; H, 6.34; N,
26
27
3
3
9
.78. Found: C, 72.88; H, 6.51; N, 9.63.
Using the similar procedure as that of 5a, starting
from 426 mg (2.0 mmol) of HCl–L-Ser-OBzl, 97 mg
(12%) of the title compound was obtained as a color-
4
zyl ester (5i)
.15. N-(1-Methyl-b-carboline-3-carbonyl)-L-proline ben-
+
less powder. Mp 158–160 ꢁC; ESI/MS: 404 [M+H] ;
IR (KBr) 3342, 3027, 3011, 1754, 1691, 1602, 1581,
Using the similar procedure as that of 5a, starting from
446 mg (2.0 mmol) of HCl–L-Pro-OBzl, 134 mg (16%) of
the title compound was obtained as a colorless powder.
ꢀ
1
1
1503, 1462, 764, 703 cm
.
HNMR (DMSO-d6)
d = 12.00 (s, 1H), 8.64 (s, 1H), 8.34 (s, 1H), 7.74 (d,
J = 8.1 Hz, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.34 (d,
J = 7.8 Hz, 2H), 7.30 (t, J = 7.5 Hz, 2H), 7.28 (t,
J = 7.5 Hz, 1H), 7.16 (t, J = 7.6 Hz, 1H), 7.03 (t,
J = 7.6 Hz, 1H), 5.27 (s, 2H), 4.72 (m, J = 5.6 Hz,
1H), 3.12 (m, J = 5.6 Hz, 2H), 2.83 (s, 3H), 2.05 (s,
+
Mp 190–191 ꢁC; ESI/MS: 414 [M+H] ; IR (KBr) 3342,
3
7
8
031, 3012, 1754, 1690, 1601, 1582, 1505, 1460, 761,
H NMR (DMSO-d ) d = 11.95 (s, 1H),
6
ꢀ
1
1
02 cm
.45 (s, 1H), 7.72 (d, J = 7.2 Hz, 1H), 7.65 (d,
.
J = 8.1 Hz, 1H), 7.31 (t, J = 7.8 Hz, 2H), 7.29 (d,
J = 7.5 Hz, 2H), 7.07 (t, J = 7.5 Hz, 1H), 7.05 (d,
J = 7.5 Hz, 1H), 5.28 (s, 2H), 4.47 (m, J = 6.6 Hz, 1H),
20
1H). ½aꢁ 4.0 (c 0.5, EtOH/DMSO, 4:1). Anal. Calcd
D
for C H N O : C, 68.47; H, 5.25; N, 10.42. Found:
C, 68.62; H, 5.44; N, 10.23.
2
3
21
3
4
3
.71 (t, J = 6.6 Hz, 2H), 2.81 (s, 3H), 2.27 (m,
20
J = 6.6 Hz, 2H), 2.02 (m, J = 6.6 Hz, 2H). ½aꢁ 2.0 (c
D
.5, EtOH/DMSO, 4:1). Anal. Calcd for C H N O :
0
4.19. N-(1-Methyl-b-carboline-3-carbonyl)-L-lysine
benzyl ester (5m)
2
5
23
3
3
C, 72.62; H, 5.61; N, 10.16. Found: C, 72.44; H, 5.43;
N, 9.95.
Using the similar procedure as that of 5a, starting from
744 mg (2.0 mmol) of HCl–Lys(Dde)-OBzl, the crude
N-(1-methyl-b-carboline-3-carbonyl)-L-lysine (Dde) benzyl
ester was directly subjected to 2% hydrazine in DMF
(5 min) to remove Dde protecting group. After concen-
tration, the residue was further purified by flash chroma-
4
.16. N-(1-Methyl-b-carboline-3-carbonyl)-L-glutamic
acid benzyl ester (5j)
Using the similar procedure as that of 5a, starting from
510 mg (2.0 mmol) of HCl–L-Glu-OBzl, 90 mg (8%) of
the title compound was obtained as a colorless powder.
Mp 211–213 ꢁC; ESI/MS: 536 [M+H] ; IR (KBr) 3340,
tography (CHCl /MeOH, 10:1) to give the title
3
+
compound as a colorless powder 71 mg (8% yield).
+
1
3
7
8
7
029, 3008, 1752, 1692, 1603, 1584, 1502, 1461, 762,
H NMR (DMSO-d ) d = 12.00 (s, 1H),
.75 (s, 1H), 8.32 (s, 1H), 7.76 (d, J = 7.8 Hz, 1H),
.60 (d, J = 7.8 Hz, 1H), 7.32 (t, J = 7.2 Hz, 2H), 7.31
ESI/MS: 445 [M+H] ; H NMR (DMSO-d ) d = 11.80
6
ꢀ
1
1
01 cm
.
(s, 1H), 8.78 (s, 1H), 8.28 (s, 1H), 7.70 (d, J = 8.0 Hz,
1H), 7.52 (d, J = 7.9 Hz, 1H), 7.34 (t, J = 7.0 Hz, 2H),
7.32 (t, J = 7.0 Hz, 2H), 7.30 (t, J = 7.6 Hz, 2H), 7.28
(t, J = 7.8 Hz, 2H), 7.26 (t, J = 7.8 Hz, 1H), 7.24 (t,
J = 7.8 Hz, 1H), 7.18 (t, J = 7.6 Hz, 1H), 7.01 (t,
J = 7.6 Hz, 1H), 5.27 (s, 2H), 4.43 (m, 1H), 2.65 (m,
2H), 2.55 (s, 3H), 1.89 (m, 2H), 1.55 (m, 2H), 1.30 (m,
6
(
t, J = 7.2 Hz, 2H), 7.30 (t, J = 7.8 Hz, 2H), 7.29 (t,
J = 7.8 Hz, 2H), 7.27 (t, J = 7.8 Hz, 1H), 7.25 (t,
J = 7.8 Hz, 1H), 7.19 (t, J = 7.6 Hz, 1H), 7.02 (t,
J = 7.6 Hz, 1H), 5.28 (s, 2H), 5.25 (s, 2H), 4.40 (m,
J = 6.6 Hz, 1H), 2.79 (s, 3H), 1.90 (m, J = 6.4 Hz, 2H),
1
3
2H). C NMR (DMSO-d ) d = 171.5, 161.7, 152.8,
6
20
1
4
.23 (t, J = 6.4 Hz, 2H). ½aꢁ 3.0 (c 0.5, EtOH/DMSO,
142.2, 140.6, 136.2, 135.4, 132.4, 129.0, 128.7, 127.1,
122.0, 120.1, 118.9, 113.4, 111.1, 105.9, 68.0, 52.8,
42.0, 32.5, 30.9, 21.0, 20.0.
D
:1). Anal. Calcd for C H N O : C, 71.76; H, 5.46;
3
2
29
3
5
N, 7.85. Found: C, 71.90; H, 5.63; N, 7.68.

7008
M. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 6998–7010
4
benzyl ester (5n)
.20. N-(1-Methyl-b-carboline-3-carbonyl)-L-arginine
d = 8.85 (s, 1H), 8.46 (s, 1H), 7.99 (s, 1H), 7.73 (d,
J = 7.5 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.12 (t,
J = 7.6 Hz, 1H), 7.01 (t, J = 7.5 Hz, 1H), 3.28 (t,
J = 4.5 Hz, 2H), 2.96 (t, J = 4.4 Hz, 2H), 2.82 (s, 3H),
2.78 (s, 2H). Anal. Calcd for C H N O: C, 67.15; H,
Using the similar procedure as that of 5a, starting from
5
28 mg (2.0 mmol) of HCl–Arg–OBzl, 94 mg (10%
yield) of the title compound purified by flash chroma-
tography was obtained as a colorless powder. ESI/MS:
1
5
16
4
6.01; N, 20.88. Found: C, 67.33; H, 6.20; N, 21.06.
+
1
+
4
8
7
73 [M+H] ; H NMR (DMSO-d ) d = 11.78 (s, 1H),
Compound 8: Mp 210–211 ꢁC; ESI/MS: 477 [M+H] .
IR (KBr) 3345, 3334, 3331, 3010, 1692, 1603, 1586,
6
.78 (s, 1H), 8.27 (s, 1H), 8.0 (m, 1H), 7.90 (m, 1H),
.70 (d, J = 8.0 Hz, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.35
ꢀ
1
1
1501, 1460, 764, 702 cm
.
H NMR (DMSO-d6)
(
t, J = 7.1 Hz, 2H), 7.33 (t, J = 7.2 Hz, 2H), 7.30 (t,
d = 8.92 (s, 2H), 8.66 (s, 2H), 8.02 (s, 2H), 7.70 (d,
J = 7.8 Hz, 2H), 7.45 (d, J = 8.1 Hz, 1H), 7.17 (t,
J = 7.5 Hz, 2H), 7.07 (t, J = 7.5 Hz, 2H), 3.32 (t,
J = 4.5 Hz, 2H), 3.02 (t, J = 4.4 Hz, 2H), 2.80 (s, 6H).
Anal. Calcd for C H N O : C, 70.57; H, 5.08; N,
J = 7.6 Hz, 2H), 7.28 (t, J = 7.6 Hz, 2H), 7.26 (t,
J = 7.6 Hz, 1H), 7.24 (t, J = 7.8 Hz, 1H), 7.18 (t,
J = 7.6 Hz, 1H), 7.01 (t, J = 7.6 Hz, 1H), 5.30 (s, 2H),
4
2
1
1
2
.42 (m, 1H), 2.66 (m, 2H), 1.89 (m, 2H), 1.55 (m,
2
8
24
6
2
1
3
H). C NMR (DMSO-d ) d = 171.6, 160.8, 158.0,
17.64. Found: C, 70.40; H, 5.27; N, 17.80.
6
51.0, 141.0, 140.5, 135.4, 135.2, 131.4, 122.2, 120.1,
19.0, 129.0, 128.0, 127.0, 113.4, 111.0, 52.6, 37.5,
5.0, 28.0, 20.0.
4.23. In vitro cytotoxicity assay
In vitro cytotoxicity assays were carried out using 96-
microtiter culture plate and MTT staining according
to the previously reported procedures with a minor
4
.21. 1-Methyl-b-carboline-3-carbonylhydrazide (6)
4
1
4
At 80 ꢁC, to the solution of 2.5 ml (50.0 mmol) of hydra-
zine hydrate in 2 ml of methanol, a mixture of 480 mg
modification. Cells (2 · 10 cells/well) were grown in
RPMI-1640 medium containing 10% (v/v) fetal calf
ꢀ
1
ꢀ1
(
2.0 mmol) of methyl 1-methyl-b-carboline-3-carboxyl-
serum and 100 lg ml penicillin and 100 lg ml strep-
tomycin. Cultures were propagated at 37 ꢁC in a humid-
ate in 10 ml of chloroform and 2 ml of methanol was
added dropwise. The reaction mixture was stirred at
ified atmosphere containing 5% CO . The synthetic
2
8
0 ꢁC for 5 h and TLC (chloroform/methanol, 15:1)
compounds’ stock solutions were prepared in DMSO.
The tumor cell line panel consists of liver carcinoma
(HepG2), cervical carcinoma (HeLa), and human breast
cancer (MCF-7). The synthetic compounds in 2%
DMSO were added to the cultures at day 1 after seeding.
After 72 h, 20 lL MTT solution (5 lg/mL) per well was
added to each culture medium and incubated for addi-
tional 4 h. The optical density was measured by the
microplate reader after adding 100 lL of 10% SDS–
0.01 N HCl solution to each well (n = 3).
indicated the complete disappearance of methyl 1-meth-
yl-b-carboline-3-carboxylate. On evaporation the resi-
due was purified by silica gel chromatography (CHCl3/
MeOH, 3:1) to provide 180 mg (38%) of the title com-
pound as a yellow powder. Mp 294–295 ꢁC; ESI/MS:
+
41 [M+H] . IR (KBr): 3340, 3335, 2924, 2910, 2900,
2
ꢀ
1
620, 1600, 1585, 1562, 1440, 1380, 1066, 900 cm ;
1
1
H NMR (DMSO-d ): d = 11.90 (s, 1H), 8.63 (s, 1H),
6
8
(
.34 (d, J = 7.8 Hz, 1H), 7.63 (d, J = 8.1 Hz, 1H), 7.56
t, J = 7.5 Hz, 1H), 7.26 (t, J = 7.5 Hz, 1H), 4.60 (m,
1
6
2
H), 2.80 (s, 3H). Anal. Calcd for C H N O : C,
12
4.99; H, 5.03; N, 23.32. Found: C, 65.18; H, 5.18; N,
3.16.
4.24. Apparent permeability coefficient test
1
3
4
2
Caco-2 cells were obtained from the American Type
Culture Collection, Rockville, MD, USA. The cells were
cultivated on polycarbonate filters (transwell cell culture
inserts, 12 mm in diameter, 3.0 lM in mean pore size) as
4
.22. N-(1-Methyl-b-carboline-3-carbonyl)-N-(aminoeth-
yl)-amine (7) and 1,2-di-(1-methyl-b-carboline-3-carbon-
yl)ethylenediamine (8)
5
1
described elsewhere. Caco-2 cells grown on filter sup-
ports for 21 days were used for all transport tests, and
the integrity of monolayers was routinely checked by
measurements of transepithelial electrical resistance
At 88 ꢁC, to 12 ml (180 mmol) of ethylenediamine, the
mixture of 1.0 g (4.2 mmol) of methyl-1-methyl-b-carb-
oline-3-carboxylate in 10 ml of methanol and 30 ml of
chloroform was added dropwise. The reaction mixture
was stirred at 88 ꢁC for 5 h until TLC (chloroform/
methanol, 15:1) indicated the complete disappearance
of methyl-1-methyl-b-carboline-3-carboxylate. On evap-
oration the residue was separated by silica gel chroma-
2
(approximately 700 X cm ).
All absorption tests were performed in Hanks’ balanced
salt solution (HBSS). Compounds 5a–n for evaluation
were dissolved in HBSS to prepare drug solutions at a
ꢀ3
final concentration of 4 · 10 M. In apical to basolater-
al direction, transport was initiated by adding drug solu-
tions (total AP volume, 0.5 ml) to the apical
compartment of inserts held in transwells containing
1.5 ml HBSS (basolateral compartment). In basolateral
to apical direction, transport was initiated by adding
tography (CHCl /MeOH, 3:1) to afford 793 mg (71%)
3
of N-(1-methyl-b-carboline-3-carbonyl)-N-(aminoeth-
yl)-amine (7) and 20 mg (1.8%) of 1,2-di-(1-methyl-b-
carboline-3-carbonyl)ethylenediamine (8) as a yellow
powder.
1
.5 ml of drug solution to basolateral compartment
+
Compound 7: Mp 172–173 ꢁC; ESI/MS: 269 [M+H] .
IR (KBr) 3341, 3332, 3330, 3009, 1690, 1600, 1585,
and adding 0.5 ml HBSS as receiving solution to apical
side of the monolayers. The monolayers were incubated
in air at 37 ꢁC and 95% relative humidity. At 30, 60, 90
ꢀ
1
1
1
500, 1464, 762, 701 cm
.
H NMR (DMSO-d6):

M. Zhao et al. / Bioorg. Med. Chem. 14 (2006) 6998–7010
7009
and 120 min, samples were withdrawn from the receiv-
ing side, and the concentrations of the samples were
determined by HPLC analysis. The resistance of mono-
layers was checked at the end of each experiment.
Apparent permeability coefficients (Papp) were calculat-
ed according to the following equation:
13. Gandolfi, C. A.; Beggiolin, G.; Menta, E.; Palumbo, M.;
Sissi, C.; Spinelli, S.; Johnson, F. J. Med. Chem. 1995, 38,
5
26–536.
1
4. Bolognese, A.; Correale, G.; Manfra, M.; Lavecchia, A.;
Mazzoni, O.; Novellino, E.; La Colla, P.; Sanna, G.;
Loddo, R. J. Med. Chem. 2004, 47, 849–858.
1
5. Lynch, M. A.; Duval, O.; Sukhanova, A.; Devy, J.;
Mackay, S. P.; Waigh, R. D.; Nabiev, I. Bioorg. Med.
Chem. Lett. 2001, 11, 2643–2646.
P
app ¼ dQ=dt ꢂ 1=ðA ꢂ C Þ
0
1
1
1
6. Nakanishi, T.; Suzuki, M.; Mashiba, A.; Ishikawa, K.;
Yokotsuka, T. J. Org. Chem. 1998, 63, 4235–4239.
7. Hayashi, K.; Nagao, M.; Sugimura, T. Nucleic Acids Res.
1977, 4, 3679–3685.
8. Meester, C. Mutat. Res. 1995, 339, 139–153.
wherein dQ/dt is the initial permeability rate, C is the
0
initial concentration in the donor chamber, and A is
the surface area of monolayer (1 cm ).
2
4
.25. Molecular modeling
19. Funayama, Y.; Nishio, K.; Wakabayashi, K.; Nagao, M.;
Shimoi, K.; Ohira, T.; Hasegawa, S.; Saijo, N. Mutat. Res.
1996, 349, 183–191.
Modeling study was performed using the Docking pro-
gram Insight II from Accelrys (version 2000, Molecular
Simulations, San Diego, CA) for structure visualization
and analysis. The AMBER force field was used in all
molecular mechanics (MM) calculations and minimiza-
tion was performed using the steepest gradient algo-
rithm with 1000 iteration steps or until convergence.
All calculations were performed on a SGI IRIX6.5
workstation.
2
2
2
0. Ishida, J.; Wang, H. K.; Bastow, K. F.; Hu, C. Q.; Lee, K.
H. Bioorg. Med. Chem. Lett. 1999, 9, 3319–3324.
1. Al-Allaf, T. A. K.; Rashan, L. J. Eur. J. Med. Chem. 1998,
33, 817–820.
2. Cao, R.; Peng, W.; Chen, H.; Ma, Y.; Liu, X.; Hou, X.;
Guan, H.; Xu, A. Biochem. Biophys. Res. Commun. 2005,
3
38, 1557–1563.
3. Remsen, J. F.; Cerutti, P. A. Biochem. Biophys. Res.
Commun. 1979, 86, 124–129.
4. Pognan, F.; Saucier, J. M.; Paoletti, C.; Kaczmarek, L.;
Nantka-Namirski, P.; Mordarski, M.; Peczynska-Czoch,
W. Biochem. Pharm. 1992, 44, 2149–2155.
2
2
Acknowledgments
2
5. Laronze, M.; Boisbrun, M.; Leonce, S.; Pfeiffer, B.;
Renard, P.; Lozach, O.; Meijer, L.; Lansiaux, A.; Bailly,
C.; Sapi, J.; Laronze, J. Y. Bioorg. Med. Chem. 2005, 13,
S.P. wishes to thank the National Natural Scientific
Foundation of China (20372007, 20232020) and
Beijing Municipal Commission of Education (KZ
2
6. Funayama, Y.; Nishio, K.; Wakabayashi, K.; Nagao, M.;
263–2283.
2
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