Benzyl 1,2,3,5,11,11a-hexahydro-3,3-dimethyl-1-oxo-6H-imidazo[3′,4′:1,2]pyridin[3,4-b]indole-2-substituted acetates: One-pot-preparation, anti-tumor activity, docking toward DNA and 3D QSAR analysis

Article abstract of DOI:10.1016/j.bmc.2010.01.038

To discover the anti-tumoral indoles a series of benzyl 1,2,3,5,11,11a-hexahydro-3,3-dimethyl-1-oxo-6H-imidazo[3′,4′:1,2]pyridin[3,4-b]indole-2-substituted acetates (2a-n) are prepared via one-pot-preparation. The IC₅₀ values of 2a-n in vitro a

Full text of DOI:10.1016/j.bmc.2010.01.038

Bioorganic & Medicinal Chemistry 18 (2010) 1910–1917
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Benzyl 1,2,3,5,11,11a-hexahydro-3,3-dimethyl-1-oxo-6H-imidazo[30,40:1,2]pyri-
din[3,4-b]indole-2-substituted acetates: One-pot-preparation, anti-tumor
activity, docking toward DNA and 3D QSAR analysis
b
a
b,
a
b
Jiawang Liu a, Ming Zhao a, , Keduo Qian , Xiaoyi Zhang , Kuo-Hsiung Lee , Jianhui Wu , Yi-Nan Liu ,
*
*
Shiqi Peng a,
*
a College of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR China
b Natural Products Research Laboratories, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7360, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
To discover the anti-tumoral indoles a series of benzyl 1,2,3,5,11,11a-hexahydro-3,3-dimethyl-1-oxo-
6H-imidazo[30,40:1,2]pyridin[3,4-b]indole-2-substituted acetates (2a–n) are prepared via one-pot-prep-
aration. The IC50 values of 2a–n in vitro against human lung carcinoma, prostate cancer, nasopharyn-
geal carcinoma, vincristine-resistant KB subline and human breast carcinoma cells range from 40 nM
Received 1 November 2009
Revised 15 January 2010
Accepted 16 January 2010
Available online 25 January 2010
to 60 lM. On Sarcoma 180 (S180) tumor-bearing mouse model four of them (2e,g,h,i) significantly
inhibited the tumor growth. At the dose of 0.1 mg/kg the efficacy of the most potent 2h was equal
to that of 1.0 mg/kg of doxorubicin. In contrast to doxorubicin, at 1.0 mg/kg of dose 2e,g,h,i did not
induce the treated S180 mice to have organ atrophy and body emaciation. The healthy mice receiving
10, 100 and 500 mg/kg of 2e,g,h,i gave no any neurotoxic response. Even up to the dose of 500 mg/kg
the healthy mice occurred no death. The interaction of 2a–n with DNA was confirmed by the fluores-
cence quenching experiments and automated flexible ligand docking. By 3D QSAR analysis the IC50
values of 2a–n against prostate cancer cells were correlated with the structures and conformations
of their side chains. To increase the data related to their physical-chemical properties the experimental
Log P values were also provided.
Keywords:
b-Carboline-3-carboxylic acid
Anti-tumor activity
Neurotoxicity
Organ damage
3D QSAR
Log P
Docking
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
topoisomerase11–13 and cyclin-dependent kinases.13,14 Among
these action mechanisms intercalation has particular importance
Despite a better understanding of the disease, the advent of
modern technology and rationally targeted drugs, the incidence
and cure rate of cancer have not improved. In the past decades con-
tinuous efforts have been made for discovering of new anti-tumor
compounds and a number of substances have been reported.
Among the discovered substances b-carboline derivatives have
been particularly known possessing diverse action mechanism. b-
Carboline derivatives may induce DNA damage,1 intercalate to-
ward DNA,2 bind minor groove,3,4 inhibit DNA synthesis5 or repair
DNA.6 b-Carboline derivatives are also capable of inducing cross-
ing-over and mitotic gene conversion7, modulating the extrinsic
or death receptor pathway and the intrinsic or mitochondrial path-
way8, as well as target Ras and their signaling pathways.9 b-Carb-
oline derivatives may also inhibit indoleamine 2,3-dioxygenase,10
in the clinical oncology and some b-carboline derivatives are rou-
tinely used.15,16
Structurally, b-carboline derivatives with DNA intercalation
activities are characterized by a polycyclic aromatic planar pharma-
cophore capable of stacking between DNA pairs.17,18 However,
DNA intercalation of substituted b-carbolines may result in serious
neurotoxicity.19 Discovering of non-neurotoxic b-carbolines has at-
tracted a lot of interests.20,21 As part of our ongoing efforts, recently
a
series of b-carboline-3- carbonylamino acid benzyl ester
conjugates were evaluated as potent anticancer agents and substi-
tuted benzyl 1,2,3,5,11,11a-hexahydro-3,3-dimethyl-1-oxo-6H-
imidazo[30,40:1,2]pyridin[3,4-b]indole-2-acetates were considered
P glucose protein (Pgp) inhibitors possessing reversing resistance
activity.22 Afterwards they were found having low neurotoxicity.
In this context, in the present paper the in vitro anti-proliferation,
in vivo anti-tumor activity, neurotoxicity, organ damage, acute tox-
icity, 3D QSAR analysis, experimental Log P values and intercalating
mechanism of b-carboline derivatives, benzyl 1,2,3,5,11,11a-hexa-
hydro-3,3-dimethyl-1-oxo-6H-imidazo[30,40:1,2]pyridin[3,4-b]in-
dole-2-substituted acetates, were investigated.
* Corresponding authors. Tel./fax: +86 10 8391 1528 (S.P.); +86 10 8280 2482
(M.Z).
E-mail addresses: mingzhao@bjmu.edu.cn (M. Zhao), shiqipeng@163.com,
sqpeng@bjmu.edu.cn (S. Peng).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.01.038

J. Liu et al. / Bioorg. Med. Chem. 18 (2010) 1910–1917
1911
Table 1
2. Results and discussion
2.1. Chemistry
IC50 values of 2a–n against A549, PC-3, MCF-7, KB and KB-VIN cell lines
Compd
IC50 (lM)
A549
PC-3
MCF-7
KB
KB-VIN
The synthetic route was depicted in Scheme 1. In this
one-pot-three-step procedure 3S-N-Boc-1,2,3,4-tetrahydro-b-carb-
oline-3-carboxylic acid was firstly prepared from the coupling of
b-carboline-3-carboxylic acid and Boc-N3. The coupling product
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
43.57
44.94
30.57
59.06
7.92
35.28
0.12
0.39
10.33
8.47
7.06
47.96
9.98
40.57
0.69
21.11
33.71
14.71
57.00
10.37
43.00
4.44
50.13
52.00
41.15
60.34
22.48
46.39
18.02
37.59
36.30
40.01
38.24
32.89
43.47
42.10
50.90
52.21
32.48
55.72
12.23
41.12
8.35
was then amidated with
L-amino acid benzylester to gave
3S-N-Boc-1,2,3,4-tetrahydro-b-carboline-3-carbonyl-L-amino acid
benzylester, which was unprotected to remove the Boc group, and
treated with acetone and triethylamine to form benzyl
1,2,3,5,11,11a-hexahydro-3,3-dimethyl-1-oxo-6H-imidazo[30,40:1,2]-
pyridin[3,4-b]indole- 2-substituted acetates (2a–n). The crystals of
2a–n were collected from methanol in 36–48% total yields. HPLC
analysis indicated that the purities of 2a–n ranged from 98.0% to
98.9%. These data suggest that this one-pot-three-step synthesis is
suitable for simply preparing 2a–n.
0.21
0.04
4.04
7.01
8.48
0.15
38.62
37.92
42.26
30.55
50.11
40.00
33.59
23.27
8.34
39.59
38.42
35.40
13.92
4.24
16.96
10.00
37.04
19.81
4.61
20.17
20.44
2.2. Effects of 2a–n on the proliferation of five human
carcinoma cell lines
Table 2
Effects of 2a–n on tumor weights of S180 micea
Compd
Tumor weight
In the anti-proliferation assays, human lung carcinoma (A549),
prostate cancer (PC-3), nasopharyngeal carcinoma (KB), vincris-
tine-resistant KB subline (KB-VIN) and human breast carcinoma
(MCF-7) cell lines were exposed to serial concentrations (60, 6,
NS
1.602 0.391
0.817 0.369b
2.846 0.330
2.003 0.284
2.050 0.447
1.418 0.075
0.916 0.370c
A 0.713 0.310d
B 0.955 0.221e
C 1.244 0.252f
1.600 0.352
1.046 0.176b
A 0.746 0.313c
B 1.013 0.207g
C 1.305 0.224h
1.578 0.498
1.793 0.147
1.613 0.360
1.604 0.295
1.696 0.156
Doxorubicin
2a
2b
2c
2d
2e
2h
0.6, 0.06 and 0.006 lM) of 2a–n, and the sulforhodamine B assays
were performed. The IC50 values of 2a–n against these five cell
lines are summarized in Table 1. The data indicate that besides
2e,g,h,i,l highly inhibit the proliferation of A549, PC-3 and MCF-7
cell lines, most of 2a–n highly inhibit the proliferation of PC-3 cell
lines. While 2a–d,f,j,k,m,n moderately inhibit the proliferation of
A549, MCF-7, KB and KB-VIN cell lines. Compounds 2e,g,h,i,l are
the most active compounds in vitro.
2f
2g
2i
2j
2k
2l
2m
2n
2.3. Effect of 2a–n on tumor weights of the treated S180 mice
The in vivo anti-tumor activities of 2a–n were evaluated on
S180 mouse model, while doxorubicin and normal saline (NS) were
used as the positive and negative controls, respectively. The data
are listed in Table 2. The data indicate that when S180 mice were
given a daily ip injection of 0.2 mL of the solution of 1.0 mg/kg of
each of 2a–n in NS for seven consecutive days 2e,g,h,i significantly
inhibit the tumor growth of S180 mice. Excepting 2l, the in vivo
determination matches with the in vitro determination. With re-
gard to the structures of 2a–n the side chain of 2l is the longest
one. Comparing to the short side chain the long one should be
more sensitive to the in vivo metabolism. This may be responsible
a
Dose of doxorubicin and 2a–n: 1.0 mg/kg; dose of 2h-A, B and C are 1.0, 0.1 and
0.01 mg/kg, respectively; NS = vehicle, n = 12, tumor weight is represented by
SD g.
ꢀ
x
b
c
d
e
f
Compared to NS p <0.01.
Compared to NS p <0.01, to 2i-B p <0.05, to doxorubicin p >0.05.
Compared to NS p <0.01, to 2h-B p <0.05, to doxorubicin p >0.05.
Compared to NS p <0.01, to 2h-C p <0.01, to doxorubicin p >0.05.
Compared to NS p <0.05.
Compared to NS p <0.01, to 2i-C p <0.05.
Compared to NS p <0.05.
g
h
O
O
O
R
OH
OH
Boc
i
ii
N
H
CO2Bzl
N
NH
N
R
N
H
Boc
O
N
N
H
H
1
H2N
CO2Bzl
iii, iv
R
N
CO2Bzl
N
N
H
2a-n
Scheme 1. Synthetic route of benzyl 1,2,3,5,11,11a-hexahydro-3,3-dimethyl-1-oxo-6H-imidazo-[30,40:1,2]pyridin[3,4-b]indole-2-substituted acetates. (i) Boc-N3, DMF; (ii)
DCC, HOBt; (iii) HCl in EtOAc(4 N); (iv) MeOH, acetone, Et3N. In 2a R = CH(CH3)CH2CH3; 2b R = CH(CH3)2; 2c R = CH2CH(CH3)2; 2d R = CH3; 2e R = H; 2f R = CH2C6H5; 2g
R = CH2C6H4-OH-p; 2h R = Indole-3-yl-CH2; 2i R = CH2CO2Bzl; 2j R = CH2CH2CO2Bzl; 2k R = CH2OBzl; 2l R = CH2CH2CH2CH2NHCBz; 2m R = CH2CONH2; 2n R = CH2CH2CONH2.

1912
J. Liu et al. / Bioorg. Med. Chem. 18 (2010) 1910–1917
for the in vivo data of 2l mismatch the in vitro data. The data also
indicate that the therapeutic potency of 2e,h,i equals to doxorubi-
cin (tumor weight, 0.817 g). This means 2e,h,i possess desirable
therapeutic efficacy.
high survival and without neurotoxic behavior implies that 2e,-
g,h,i should be promising anti-tumor leads.
2.6. Log K of 2a–n
The most in vivo active 2h and 2i were observed at doses of 1.0,
0.1 and 0.01 mg/kg to produce a possible dose-dependent in vivo
anti-tumor response in S180 mice. The data are also listed in Table
2, which demonstrate that the tumor weight of the treated mice is
progressively increased with dose decrease. Therefore, these
experiments explored that ip injection of 2h and 2i exhibited
dose-dependent anti-tumor action.
The Log P value, defined as the logarithm of the partition coeffi-
cient between n-octanol and water, is an important parameter to
reflect a compound’s bioavailability. Meanwhile, it has been widely
used as an important molecular descriptor in QSAR such as the
classical Hansch approach.23 It is well known that Log K values
are highly correlated with the corresponding Log P values, and
have been generally used to replace the time-consuming Log P
determination. Therefore, the Log K values of 2a–n provided here
could be practically useful.
2.4. Organ and body damage of 2e,g,h,i to the treated S180 mice
As one of visible results of chemotherapeutic toxicity related or-
gan damage should be organ atrophy. To evaluate the organ and
body damage the organ and body weights of the most potency
2e,g,h,i treated S180 mice were measured. Twenty-four hours after
the last administration the mice were measured for body weights,
and then all mice were sacrificed by diethyl ether anesthesia and
dissected to immediately obtain and weigh the liver, kidney, brain,
spleen, heart and left femur samples (Table 3). It was found that
except brain, the weights of the liver, kidney, spleen, heart, left fe-
mur and body of doxorubicin receiving S180 mice were signifi-
cantly lower than that of NS receiving S180 mice. This
comparison demonstrates that doxorubicin therapy may induce
organ atrophy and body emaciation for S180 mice. While the
weights of the liver, kidney, spleen, heart, left femur and body of
2e,g,h,i receiving S180 mice were not significantly different from
that of NS receiving S180 mice. This comparison demonstrates that
2e,g,h,i therapy results no similar side effects to that of
doxorubicin.
Each of 2a–n was dissolved in aqueous methanol (50%) to pre-
pare sample solution (10
on Agilent 1100 series, the column was a reversed-phase C18 col-
umn (Agilent Zorbax Extend-C18, 4.6 ꢀ 15 mm, 5 m). After 20
lM). The HPLC analysis was carried out
l
lL
of the sample solution was loaded, the column was eluted with
50% solution of MeOH as the mobile phase for 40 min. The flow
rate was 1 mL/min. Each of 2a–n in the sample was monitored
with UV detector at 254.8 nm and the retention time (tR) of the
peak was recorded. In the same HPLC conditions the retention time
of acetone peak was recorded as t0. In order to offset the influence
of the solvent on the appearance time of the peak of 2a–n, the
appearance time of acetone peak (t0, 1.591 min) was used as a con-
trol. According to the common knowledge that Log K is alternative
representation of tR, the Log K values of 2a–n were calculated
based on the equation Log K = Log[(tR ꢁ t0)/t0] and are listed in Ta-
ble 4.
It was noticed that the Log K values of 2a–n not correlated with
the substituent hydrophobicity constant. By using the QSAR mod-
ule of Cerius2 the substituent hydrophobicity constant based Log P
values were calculated and are also displayed in Table 4. To discuss
the correlation of the Log K value with the experimental Log P val-
ues the calibration curves of 2a–n are making. The experimental
Log P values, the correlation of the Log K value with the experimen-
tal Log P value and the QSAR analysis of the experimental Log P va-
lue and anti-tumor activity will be reported in a separate paper.
2.5. Neurotoxic and acute toxicities of 2e,g,h,i receiving healthy
mice
Neurotoxic and acute toxicities of the most potent 2e,g,h,i re-
ceived healthy mice were assayed by use of a general procedure.
It was found that during 7-day observation 10, 100 and 500 mg/
kg of 2e,g,h,i (ip) receiving healthy mice exhibited no any neuro-
toxic behavior, such as tremor, twitch, jumping, tetanus, as well
as supination. On the seventh day the necropsy findings of the
mice gave also no apparent changes in their organs. These observa-
tions suggest that 2e,g,h,i should be low toxic agents. Besides, even
up to 500 mg/kg of dose the treated healthy mice occurred no
death. This observation suggests that the LD50 value of 2e,g,h,i
are more than 500 mg/kg. It is generally accepted that most of b-
carbolines are strong neurotoxicity and the treated animals have
low survival.22 At the dose of 500 mg/kg the treated mice having
2.7. 3D QSAR equation of 2a–n inhibiting proliferation of PC-3
cell lines
To analyze the relationship of the structures and the anti-tumor
activities of 2a–n the valid 3D QSAR models of Cerius2 were used.
With an assumption that all structures of 2a–n bind the same site
of the receptor with their common hexahydro-3,3-dimethyl-1-
oxo-6H-imidazo[30,40:1,2]pyridin[3,4-b]indole ring the maximum
common subgraph (MCS) was used.24 After energy-minimization
Table 3
Body and organ weights of 2e,g,h,i receiving S180 micea
Compd
Body and organ weight (mean SD g)
Body
Brain
Heart
Liver
Spleen
Kidney
Femur
NS
DXB
2e
2g
2h
2i
33.30 2.32
27.28 2.40b
33.75 1.52
35.85 3.03
32.89 3.79
33.37 2.19
0.31 0.01
0.32 0.01
0.30 0.02
0.32 0.01
0.30 0.02
0.31 0.01
0.17 0.01
0.15 0.03c
0.16 0.02
0.16 0.01
0.17 0.02
0.17 0.03
2.36 0.20
2.00 0.25d
2.25 0.30
2.35 0.22
2.29 0.25
2.26 0.21
0.22 0.04
0.11 0.02b
0.24 0.03
0.23 0.02
0.22 0.03
0.24 0.02
0.23 0.02
0.19 0.01e
0.22 0.02
0.23 0.02
0.21 0.02
0.23 0.01
0.056 0.006
0.046 0.003b
0.055 0.004
0.056 0.005
0.050 0.003
0.057 0.005
a
b
c
Dose of doxorubicin and 2e,g,h,i: 1.0 mg/kg, NS = vehicle, n = 12.
Compare to NS and 2e,g,h,i p <0.01.
Compare to NS p <0.05.
Compare to NS and 2g,h p <0.01, to 2e,i p <0.05.
Compare to NS and 2e,g,h,i p <0.05.
d
e

J. Liu et al. / Bioorg. Med. Chem. 18 (2010) 1910–1917
1913
Table 4
positions large groups will increase the activity. The terms of
1.25 (HOꢁ/587) and 0.42 (HOꢁ/885) have positive coefficients,
which means that at these positions hydrogen bond forming
groups will decrease the activity, while the terms of 0.46 (HOꢁ/
578), 0.055 (HOꢁ/584), 0.24 (HOꢁ/797) and 0.09 (HOꢁ/918) have
negative coefficients, which means that at these positions hydro-
gen bond forming groups will increase the activity. These struc-
tural requirements could be useful for the design of novel
compounds.
Log K and Calculated Log P values of 2a–n
Compd
Log K
Cal. Log P
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
0.80
0.73
0.92
0.44
0.32
1.05
0.49
0.81
0.96
1.05
1.02
0.87
0.16
0.25
2.79
2.26
2.79
1.33
0.93
3.05
2.85
2.41
0.30
2.8. Diagrammatic examples of Eq. 1
0.83
2.00
3.15
Figure 2 gives 2h,i as diagrammatic examples of Eq. 1. Com-
pound 2h has small group near the region of CH3/798, as well as
hydrogen bond forming groups near the regions of HOꢁ/578, HO
ꢁ/797 and HOꢁ/918 thus it has high in vitro anti-proliferation
ꢁ0.105
ꢁ0.522
activity. Using Eq. 1 the IC50 of 2h was calculated to be 0.14 lM.
Compound 2i has small group near the regions of CH3/798, as well
as a hydrogen bond forming groups near the regions of HOꢁ/578,
HOꢁ/584, HOꢁ/797 and HOꢁ/918, thus it also has high in vitro
anti-proliferation activity. Using Eq. 1 the IC50 of 2i was calculated
using the MMFF94 (Merck Molecular Force Field), the Molecular
Field Analysis (MFA) was performed for 2a–n using the QSAR mod-
ule of Cerius2.25 Three probes were chosen to describe 2a–n.
Methyl group was used to account for steric contacts, while proton
and hydroxyl ion probes were used to evaluate electrostatic poten-
tial fields. The model of MFA of 2a–n inhibiting the proliferation of
PC-3 cell lines in terms of the descriptors proton, methyl and hy-
droxyl ion was expressed by Eq. 1. The data points (n), correlation
coefficient (r) and square correlation coefficient (r2) of Eq. 1 were
14, 0.996 and 0.991, respectively. The IC50 values of 2a–n and their
analogs against the proliferation of PC-3 cell lines can be predicted
with Eq. 1. With the predicted IC50 values as the ordinate and the
experimental IC50 values as the abscissa Figure 1 was obtained.
to be 0.22 lM.
Figure 3 gives 2d,f as diagrammatic examples of Eq. 1. Com-
pound 2d has electron-releasing group near the region of H+/478,
small groups near the regions of CH3/437 and CH3/783, as well as
hydrogen bond forming groups near the regions of HOꢁ/587 and
HOꢁ/885, thus it has low in vitro anti-proliferation activity. Using
Eq. 1 the IC50 of 2d was calculated to be 47.3 lM. Compound 2f
has electron-releasing group near the region of H+/478, small
groups near the regions of CH3/437, CH3/783 and CH3/928, as well
as a hydrogen bond forming group near the region of HOꢁ/587,
thus it also has low in vitro anti-proliferation activity. Using Eq.
IC50 ¼ 10:26 ꢁ 0:30ðHþ=478Þ ꢁ 0:9ðCH3=437Þ
ꢁ 0:028ðCH3=783Þ þ 0:46ðCH3=798Þ ꢁ 0:08ðCH3=928Þ
ꢁ 0:46ðHOꢁ=578Þ ꢁ 0:055ðHOꢁ=584Þ
1 the IC50 of 2f was calculated to be 40.9 lM.
2.9. Fluorescence evidence for the interaction of 2h,i with DNA
þ 1:25ðHOꢁ=587Þ ꢁ 0:24ðHOꢁ=797Þ þ 0:42ðHOꢁ=885Þ
ꢁ 0:09ðHOꢁ=918Þ
ð1Þ
DNA spectra are widely used to study the interaction of small
molecules with DNA. To reveal the possible interaction CT DNA
was selected as the model DNA, and the interaction of 2a–n with
DNA was explained by fluorescence tests of the system of CT DNA
and the most potent 2h and 2i. The effect of CT DNA on the fluores-
cence intensity of 2h or 2i was determined on fluorescence spec-
Eq. 1 contains 1 term from proton descriptor, 4 terms from
methyl descriptor and 6 terms from hydroxyl anion descriptor.
The term of 0.30 (H+/478) has negative coefficient, which means
that at this position electron-withdrawing group will increase
the activity. The term of 0.46 (CH3/798) has positive coefficient,
which means that at this position small group will increase the
activity, while terms of 0.9 (CH3/437), 0.028 (CH3/783), and 0.08
(CH3/928) have negative coefficients, which means that at these
troscopy at 300 K. The solution of 2h or 2i in PBS (1.0
was titrated with 10 L of the solution of CT DNA in PBS at a series
of concentrations (0, 9, 18, 27, 36, 45, 54, 63, 72, 81 M, pH 7.4) to
lM, pH 7.4)
l
l
observe the change of the fluorescence intensity on a Shimadzu RF-
5310PC spectrofluorometer at a fluorescence excitation wavelength
of 245 nm. It was found that CT DNA titration defined a concentra-
tion-dependent decrease of fluorescence intensities (fluorescence
quenching) of 2h and 2i. Figure 4 describes a typical course of the
fluorescence quenching, that is, a gradual decrease of the fluores-
cence intensity of 2h and 2i follows a gradual increase of the con-
centration of CT DNA. When the concentration of CT DNA was
increased to 81
ered to their minima. The fluorescence quenching of 2h and 2i in-
duced by 81 M of CT DNA was 45.07%. From the courses of the
lM the fluorescence intensity of 2h and 2i was low-
l
fluorescence quenching a set of slightly bathochromic shift was no-
ticed. This bathochromic shift is considered associating with the de-
crease in the energy gap between the highest occupied molecular
orbital and the lowest occupied molecular orbital occurs in the
interaction of 2a–n with CT DNA.21,26
2.10. Docking of 2a–n toward oligoribonucleotides
For the docking investigation of drugs toward DNA 263D (minor/
major groove binding), 1NAB-2 (intercalation plus groove binding)
Figure 1. Graph of tested versus predicted activities of 2a–n inhibiting the
proliferation of PC-3 cells.

1914
J. Liu et al. / Bioorg. Med. Chem. 18 (2010) 1910–1917
Figure 2. Electrostatic and environments of 2h (a) and 2i (b) within the grid with 3D points of Eq. 1.
Figure 3. Electrostatic and environments of 2d (a) and 2f (b) within the grid with 3D points of Eq. 1.
Figure 4. Fluorescent spectra of 2h,i (concentration 1 lM, pH 7.4, kmax = 254 nm) with serial concentrations (0, 9, 18, 27, 36, 45, 54, 63, 72, 81 lM) of CT DNA in PBS.
and 1NAB-1 (intercalation) have been considered, and the double-
stranded oligoribonucleotides d(CGATCG)2 (PDB ID: 1NAB),27
as well as d(CGCAAATTTGCG)2 (PDB ID: 263D),28 have been used
to simulate DNA. To explore the interaction patterns of 2a–n with
DNA, based on the generalized CHARMm force field in Discovery
Studio Modeling 2.1, the automated docking studies were per-
docking energies of 2a–n were calculated and the data are listed in
Table 5.
The final docking energy indicates that though there are three
interaction patterns, 2a–n are able to take two of them only. For in-
stance, 2a–g,m,n possibly take 1NAB-1 and 263D, 2i–l possibly
take 1NAB-1 and 1NAB-2, while 2h possibly take 1NAB-2 and
263D. On the other hand, for the most potent 2e, 2g, 2h and 2i
the orders of the final docking energies are 263D < 1NAB-
formed. In these studies a standard procedure of the LIGAND-FIT
/LIGAND-
SCORE was followed. Following the three interaction patterns the final

J. Liu et al. / Bioorg. Med. Chem. 18 (2010) 1910–1917
1915
Table 5
4. Experimental section
4.1. Synthesis
Final docking energy (kcal/mol) of 2a–n toward DNA
Compd
1NAB
263D
1NAB-1
1NAB-2
4.1.1. General
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
ꢁ45.60
ꢁ27.00
ꢁ75.57
ꢁ26.83
ꢁ77.17
ꢁ57.20
ꢁ0.50
—
—
—
—
—
—
—
ꢁ68.00
ꢁ53.44
ꢁ60.47
ꢁ20.80
ꢁ118.45
ꢁ3.90
ꢁ104.80
ꢁ44.10
—
The protected amino acids with
L
-configuration were purchased
from Sigma Chemical Co. All the coupling and deprotective reac-
tions were carried out under anhydrous conditions. Chromatogra-
phy was performed on Qingdao silica gel H. The purities of the
intermediates and the products were tested on TLC (Merck silica
gel plates of type 60 F254
, 0.25 mm layer thickness) and
—
ꢁ73.12
ꢁ23.13
ꢁ29.18
ꢁ77.64
ꢁ59.63
—
ꢁ89.83
ꢁ54.79
ꢁ31.40
ꢁ34.00
ꢁ73.05
ꢁ56.00
HPLC (Waters, C18 column 4.6 ꢀ 150 mm), respectively. HPLC puri-
ties of 1,2,3,5,11,11a-hexahydro-3,3-dimethyl-1-oxo-6H-imidazo-
[30,40:1,2]pyridin[3,4-b]indole-2-substituted acetates (2a–n) range
from 98.0% to 98.9%. 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 date was carried out
by use of ANOVA test with p <0.05 as significant cut-off.
—
—
—
ꢁ43.95
—
ꢁ33.76
1 < 1NAB-2, 263D < 1NAB-1 < 1NAB-2, 1NAB-2 < 263D < 1NAB-1
and 1NAB-1 < 1NAB-2 < 263D, respectively. This means that 2e,
2g, 2h and 2i may interact with DNA by selecting 263D, 1NAB-2
4.1.2. 3S-1,2,3,4-Tetrahydro-b-carboline-3-carboxylic acid (1)
and 1NAB-1, respectively.
A selective pattern matches the
To a mixture of 5.0 g (24.5 mmol) of L-tryptophane, 25 mL of
lowest docking energy. To visualize the interaction pattern the
graphical descriptions of the selective docking of 2e,g,h,i toward
oligoribonucleotides were provided (Fig. 5). Compounds 2e, 2g,
2h and 2i have individual interaction pattern implies that a
change in the structure may result in the alteration of interaction
pattern.
H2SO4 (1 mol/L) and 80 mL of water 8 mL of formaldehyde (36–
38%) was added. The reaction mixture was stirred at room temper-
ature for 2 h and adjusted to pH 6–7 with concentrated ammonia
liquor. The mixture obtained was kept at 0 °C for 12 h and the
formed precipitates were collected by filtration. After recrystalliza-
tion 3.97 g (75%) of the title compound were obtained as a colorless
powder. Mp 280–282 °C; ESI/MS (m/z) 217 [M+H]+; IR (KBr): 3450,
3. Conclusions
3200, 3000, 2950, 2850, 1700, 1601, 1452, 1070, 900 cmꢁ1 1H
;
NMR (BHSC-500, DMSO-d6): d = 10.99 (s, 1H), 9.89 (s, 1H), 7.30
(t, J = 7.5 Hz, 1H), 7.22 (t, J = 8.0 Hz, 1H), 7.01 (d, J = 8.0 Hz, 1H),
6.81 (d, J = 7.5 Hz, 1H), 4.01 (t, J = 4.8 Hz, 1H), 3.75 (dd,
J = 10.5 Hz, J = 5.0 Hz, 1H), 3.64 (dd, J = 10.5 Hz, J = 2.4 Hz, 1H),
2.91 (d, J = 10.5 Hz, 2H), 2.86 (s, 1H).
In a previous paper 2a–n were prepared by following a 4-step-
route in 22–33% total yields. In this paper 2a–n were preparedby fol-
lowing a one-pot-preparation in 36–48% total yields. Thus the prep-
aration of 2a–n was improved. In vitro some of the present
compounds were capable of inhibiting the proliferation of cancer
cell lines at a nanomolar concentration. In the toxicology evaluation
the healthy mice receiving up to a dose of 500 mg/kg of some of the
present compounds neither gave neurotoxic response nor occurred
death. In vivo even 0.01 mg/kg of 2h and 2i were still able to inhibit
the tumor growth of the treated S180 mice. The fluorescence titra-
tion indicated that 2a–n were able to interact with DNA. In the oli-
goribonucleotides based docking studies 2a–n showed individual
preferring interaction pattern. Combining the similar in vitro and
in vivo anti-tumor activities of 2e,g,h,i with their individual prefer-
ring interaction pattern it is demonstrated that they may gain their
in vitro and in vivo anti-tumor action via individual interaction
pattern.
4.1.3. General procedure for the preparation of benzyl
1,2,3,5,11,11a-hexahydro-3,3-dimethyl-1-oxo-6H-
imidazo[30,40:1,2]pyridin[3,4-b]indole-2-substitutedacetate
(2a–n)
(A) The suspension of 550 mg (2.5 mmol) of 1 in 10 mL of DMF
and 0.8 mL of triethylamine was vigorously stirred at room
temperature, to which 376 mg (2.6 mmol) of Boc-N3 was added
in 30 min. The reaction mixture was stirred at room temperature
for 12 h and at 40 °C for 20 h and TLC (CHCl3/CH3OH, 5:1) indicated
complete disappearance of 1. The reaction mixture was
cooled to 0 °C.
Figure 5. Stereoview of the docking complexes of 2e/263D, 2g/263D, 2h/1NAB-2 and 2i/1NAB-1.

1916
J. Liu et al. / Bioorg. Med. Chem. 18 (2010) 1910–1917
(B) To this reaction mixture 10 mL of anhydrous THF and
(%) = [(A ꢁ B)/A]ꢀ100, wherein A is average tumor weight of the
negative control, and B is that of the treatment group.
377 mg (2.8 mmol) of HOBt were added, after 10 min 556 mg
(2.7 mmol) of DCC were added, after another 10 min a solution of
2.6 mmol of amino acid benzylester in 3 mL of anhydrous THF
were then added. The reaction mixture was stirred at 0 °C for 2 h
and at room temperature for 8 h.
(C) On evaporation the residue was dissolved in 5 mL of anhy-
drous ethyl acetate and the solution was cooled to 0 °C and fil-
trated. To the filtrate 16 mL of hydrogen chloride/ethyl acetate
(4 N) were added dropwise. The reaction solution was stirred at
0 °C for 90 min and evaporated under reduced pressure. The resi-
due obtained was dissolved in 50 mL of methanol and 10 mL of
acetone. With triethylamine the reaction solution was adjusted
to pH 9, at room temperature and in dark stirred for 240 h, and
TLC (CHCl3/CH3OH, 10/1) indicated the complete disappearance
4.2.3. Acute toxicities assay
ICR mice, purchased from Peking University Health Science Cen-
ter, were maintained at 21 °C with a natural day/night cycle in a
conventional animal colony. The mice were 10–12 weeks old at
the beginning of the experiments. The sterile food and water were
provided according to institutional guidelines. Prior to each exper-
iment, mice were fastened overnight and allowed free access to
water. The mice were given an ip injection of 10, 100 or 500 mg/
kg of 2e,g,h,i in 0.2 mL of 0.9% saline. Each group contained 12
mice (six males and six females). After the administration the mice
were monitored continuously for up to 7 days to observe any
abnormal behavior or death. All animals were sacrificed on the sev-
enth day and checked macroscopically for possible damage to the
heart, liver, and kidneys. LD50 values were calculated graphically.
The study described herein was performed according to a protocol
reviewed and approved by the ethics committee of Capital Medical
University. The committee ensures that the welfare of the animals
is maintained in accordance to the requirements of the Animal
Welfare Act and according to the Guide for Care and Use of Labo-
ratory Animals.
of N-[(3S)-N-1,2,3,4-tetrahydro-b-carboline-3-carbonyl]-L-amino
acid benzylester. On evaporation the residue was dissolved in
5 mL of methanol to give 2a–n as crystals (Analytical data see Sup-
plementary data).
4.2. Bioassays
4.2.1. In vitro cytotoxic assay
All stock cultures were grown in T-25 flasks. Freshly trypsinizer
cell suspensions were seeded in 96-well microtiter plates at densi-
ties of 5000 cells per well with 2a–n added from DMSO-diluted
stock. After 3 days in culture, attached cells were fixed with cold
10% trichloroacetic acid and then stained with 0.4% sulforhoda-
mine B. The absorbance at 562 nm was measured using a micro-
plate reader after solubilizing the bound dye. The mean IC50 is
the concentration of 2a–n that reduces cell growth by 50% under
the experimental conditions and is the average from at least three
independent determinations that were reproducible and statisti-
cally significant. The following human tumor cell lines were used
in the assay: A549, PC-3, KB, KB-VIN, and MCF-7. All cell lines were
obtained either from the Lineberger Cancer Center (UNC-CH) or
from ATCC (Rockvile, MD) and were cultured in RPMI-1640 med-
ium supplemented with 25 mM HEPES, 0.25% sodium bicarbonate,
4.3. Determination of the retention time and thus Log K of
2a–n
Benzyl 1,2,3,5,11,11a-Hexahydro-3,3-dimethyl-1-oxo-6H-imi-
dazo[30,40:1,2]pyridin[3,4-b]indole-2-substitutedacetates 2a–n
were dissolved in aqueous methanol (50%) to prepare sample solu-
tions of 10
series, the column was a reversed-phase C18 column (Agilent 1100
m). After 20
lM. The HPLC analysis was carried out on Agilent 1100
series, Agilent Zorbax Extend-C18, 4.6 ꢀ 15 mm, 5
l
lL
of the sample (10ꢁ5 M) was loaded, the column was eluted with a
solution consisted of 0.7 CH3OH/0.3 H2O as the mobile phase for
40 min. The flow rate was 1 mL/min. The peak of 2a–n in the sam-
ple was monitored with UV detector at 254.8 nm and the retention
time (tR) corresponding to its peak was recorded. With the same
HPLC conditions the retention time of acetone peak was recorded
as t0. In order to offset the influence of the solvent on the
appearance time of the peak of 2a–n, the appearance time of ace-
tone peak (t0, 1.591 min) was used as a control. As an alternative
representation of tR, Log K was defined based on the equation
Log K = Log[(tR ꢁ t0)/t0].
10% fetal bovine serum, and 100 lg/mL kanamycin.
4.2.2. In vivo anti-tumor assay
Male ICR mice, purchased from Peking University Health Sci-
ence Center, were maintained at 21 °C with a natural day/night cy-
cle in a conventional animal colony. The mice were 10–12 weeks
old at the beginning of the experiments. S180 ascites tumor cells
were used to form solid tumors after subcutaneously injection.
For initiation of subcutaneous tumors the cells were obtained as
an ascitic form from the tumor-bearing mice, which were serially
transplanted once per week. Subcutaneous tumors were implanted
by injecting 0.2 mL of 0.9% saline containing 1 ꢀ 107 viable tumor
cells under the skin on the right armpit. Twenty-four hours after
implantation, the mice (twelve per group) were randomly divided
into experimental groups. Doxorubicin is well known as an interca-
lating agent. To clarify the validity of the mouse model and to have
a similar action mechanism doxorubicin was selected as the posi-
tive control. The mice of the positive control group were given a
daily ip injection of 1.0 mg/kg of doxorubicin in 0.2 mL of 0.9% sal-
ine for seven consecutive days. The mice of the negative control
group were given a daily ip injection of 0.2 mL of 0.9% saline for se-
ven consecutive days. The mice of the treatment groups were given
a daily ip injection of 1.0 mg/kg of 2a–n in 0.2 mL of 0.9% saline for
seven consecutive days. The weights of animals were recorded
everyday. Twenty-four hours after the last administration, all mice
were weighed, sacrificed by diethyl ether anesthesia and dissected
to immediately obtain and weigh the tumor and spleen samples.
The inhibition ratio was calculated based on inhibition ratio
Acknowledgements
This work was finished in Beijing Area Major Laboratory of
Peptide and Small Molecular Drugs, supported by the National
Natural Scientific Foundation of China (30801426, 30901843)
Special Project (2008ZX09401-002) of China and PHR (IHLB,
KZ200810025010) and PHR (IHLB, KZ200810025010
KZ200910025004).
&
Supplementary data
Supplementary data (analytical data (1H, 13C NMR, MS, etc.) for
compounds 2a–n) associated with this article can be found, in the
online version, at doi:10.1016/j.bmc.2010.01.038.
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