A class of novel N-isoquinoline-3-carbonyl-l-amino acid benzylesters: Synthesis, anti-tumor evaluation and 3D QSAR analysis

Article abstract of DOI:10.1016/j.ejmech.2011.02.017

Isoquinoline-3-carboxylic acid (2) was modified with amino acid benzylesters and 18 novel N-isoquinoline-3-carbonylamino acid benzylesters (3a-r) were provided. The IC₅₀ values of 3a-r against the proliferation of HL-60 and Hela cells were less than 1 × 10^-8 M and 6 × 10^-7 M, respectively. On S180 mice model 100 μmol/kg of 3a-r effectively inhibited the growth of the tumors. Using MFA based Cerius² QSAR module, two equations (r, 0.989 and 0.987) were established to correlate the structure with the in vitro and in vivo activities. The benefit of this modification was supported with both the in vitro membrane permeation test and the in vivo anti-tumor assay. The in vitro membrane permeability of N-isoquinoline-3-carbonyl-l-threonine benzylester (3n) and N-isoquinoline-3-carbonyl-l-leucine benzylester (3q) was 2.5 fold higher than that of 2, and the in vivo anti-tumor activity of 3n, q was 4.4-fold higher than that of 2.

Full text of DOI:10.1016/j.ejmech.2011.02.017

European Journal of Medicinal Chemistry 46 (2011) 1672e1681
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A class of novel N-isoquinoline-3-carbonyl- -amino acid benzylesters: Synthesis,
L
anti-tumor evaluation and 3D QSAR analysis
,
a
a
b,
a
Meiqing Zheng a, Yifan Yang a, Ming Zhao a , Xiaoyi Zhang , Jianhui Wu , Gong Chen
, Li Peng ,
**
***
,
Yuji Wang a, Shiqi Peng a
*
a College of Pharmaceutical Sciences, Capital Medical University, No. 10, Xitoutiao, You An Men, Beijing 100069, PR China
b Department of chemistry, The Pennsylvania state University, 104 chemistry Building, University Park, PA 16802, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Isoquinoline-3-carboxylic acid (2) was modified with amino acid benzylesters and 18 novel N-iso-
quinoline-3-carbonylamino acid benzylesters (3aer) were provided. The IC50 values of 3aer against the
proliferation of HL-60 and Hela cells were less than 1 ꢀ 10ꢁ8 M and 6 ꢀ 10ꢁ7 M, respectively. On S180
Received 29 July 2010
Received in revised form
3 January 2011
Accepted 12 February 2011
Available online 22 February 2011
mice model 100 m
mol/kg of 3aer effectively inhibited the growth of the tumors. Using MFA based Cerius2
QSAR module, two equations (r, 0.989 and 0.987) were established to correlate the structure with the in
vitro and in vivo activities. The benefit of this modification was supported with both the in vitro
membrane permeation test and the in vivo anti-tumor assay. The in vitro membrane permeability of N-
isoquinoline-3-carbonyl-L-threonine benzylester (3n) and N-isoquinoline-3-carbonyl-L-leucine benzy-
lester (3q) was 2.5 fold higher than that of 2, and the in vivo anti-tumor activity of 3n, q was 4.4-fold
higher than that of 2.
Keywords:
N-Isoquinoline-3-carbonylamino acid
benzylester
Intercalator
Ó 2011 Elsevier Masson SAS. All rights reserved.
Anti-tumor
3D QSAR
Membrane permeability
1. Introduction
kinase-b [29], caspase-3 [30], and mammalian sterile 20 kinase
[31]. Alternately, via alkylation toward the minor groove of DNA,
generating minimum intermediates or intercalating into the double
strands of the DNA isoquinoline derivatives can directly act on the
DNA of cancer cells [32e39]. On the other hand, isoquinoline-3-
carboxylic acid and derivatives have been explored to be the rigid
mimic of tyrosine conformation in the opioid ligandereceptor
complex [40], the reverse turn mimetics [41], the antagonists of
ampa receptor [42], the inhibitors on the binding of IGF to IGF-
binding proteins [43], and the antagonists of AT1 receptor [44].
However, so far no anti-tumoral isoquinoline-3-carboxylic acid
derivatives were disclosed. Due to our ongoing interest in the
discovery of isoquinolines functioning in the therapy of leukemia or
cervical carcinoma, the structural correlation of isoquinoline-3-
In spite of the total progress in diagnosis and therapy, cancer is
still one of the major human diseases and causes great suffering
and financial loss worldwide. To improve the chemotherapy in the
past decades continuous efforts have been made for discovering
new anti-tumoral leads and a great diversity of substances has been
explored. Among the discovered substances, isoquinoline deriva-
tives are capable of acting on cell-cycle [1e3] and farne-
soid ꢀ receptor [4], inhibit IGF to IGF-binding proteins [5], function
as redox-cycler [6e8], and are involved in the mutant B-Raf
pathway and the pathway that relate to extracellular signal-regu-
lated kinase/c-Jun N-terminal kinase [9,10]. Besides, isoquinoline
derivatives are the inhibitors of a number of cancer related
enzymes including inosine 50-monophosphate dehydrogenase [11],
Pfmrk [12], P-glycoprotein [13e15], kinase B/Akt [16], cyclin-
dependent kinase 4 [17], topoisomerase I [18e27], TRPV1 [28], IkB
carboxylic acid with 1-methyl-
increase of the in vitro anti-proliferation activity resulted from the
structure-simplification from 1-methyl- -carboline-3-carboxylic
acid to isoquinoline-3-carboxylic acid (Fig. 1), and the benzyl ester
of 1-methyl- -carboline-3-carboxylic acid having higher in vitro
b-carboline-3-carboxylic acid, the
b
b
anti-proliferation activity than the acid and methyl ester [45,46],
this paper describes the preparation, the in vitro anti-proliferation
of HL-60 and Hela cell lines, the in vivo anti-tumor activities, and
the 3D QSAR analysis of 18 novel N-isoquinoline-3-carbonyl-L-
amino acid benzylesters.
* Corresponding author. Tel./fax: þ86 10 8391 1528.
** Corresponding author. Tel./fax: þ86 10 8280 2482.
*** Corresponding author. Tel.: þ1 (814) 867 2590; fax: þ1 (814) 863 5319.
E-mail addresses: mingzhao@mail.bjmu.edu.cn (M. Zhao), guc11@psu.edu
(G. Chen), sqpeng@mail.bjmu.edu.cn (S. Peng).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.02.017

M. Zheng et al. / European Journal of Medicinal Chemistry 46 (2011) 1672e1681
1673
determined. The results are listed in Table 1. The IC50 values of 3aer
inhibiting the growth of HL-60 cells ranged from 44.50 to 99.94 nM,
while the IC50 values of 3aer inhibiting the growth of Hela cells
ranged from 87.74 to 507.94 nM. It was explored that for both HL-60
and Hela cells 3b,e,g,n,q consistently exhibited desirable inhibition
activity.
O
O
OH
OH
N
N
N
H
CH3
140
180
HepG2 189
Hela
MCF-7
1.32
2.3. In vivo anti-tumor activity of 3aer
2.41
2.65
IC50 (µM)
Following a general procedure the in vivo assay was carried out.
In the assays the tumor weight of the treated S180 mice was used to
Fig. 1. Structural correlation and comparison of the in vitro anti-proliferation activities
of -carboline-3-carboxylic acid (data from Ref. [45]) and isoquinoline-3-carboxylic
acid (data from the preliminary assays of this paper).
express the activity of 3aer (100
0.2 ml of normal saline (NS) as the negative control and cytarabine
(100 mol/kg/day in 0.2 ml of NS) as the positive control. The
mmol/kg/day in 0.2 ml of NS) with
b
m
intraperitoneal treatment was carried out for 7 consecutive days.
The results are listed in Table 2. The data indicate that the tumor
weights of 3aer treated mice range from 0.821 to 1.588 g and are
significantly lower than that of NS treated mice (1.802 g, p < 0.01),
which suggests that at this dose they possess anti-tumor activity. In
2. Results and discussion
2.1. Synthesis of N-isoquinoline-3-carbonyl-L-amino acid
benzylesters 3aer
addition, the anti-tumor efficacy of 100
mmol/kg/day of 3n, q is
The preparation of N-isoquinoline-3-carbonylamino acid ben-
zylesters (3aer) was carried out according to the three-step-route
of Scheme 1. In the first step, via PicteteSpengler condensation L-
Phe was converted into 3S-1,2,3,4-tetrahydroisoquinoline-3-
carboxylic acid (1, 84% yield). In the second step, 1 was oxidized
with KMnO4 and isoquinoline-3-carboxylic acid 2 was obtained in
83% yield. In the third step, amino acid benzylesters were intro-
substantially equal to that of 100 mol/kg/day of cytarabine
m
(0.814 g, p > 0.05), and 3a, e, g exhibit desirable in vivo anti-tumor
efficacy. Therefore in both in vitro and in vivo assays 3e, g, n, q
consistently give desirable activity. It was also noticed that the in
vitro efficacy did not match the in vivo efficacy. It has been well
known that the in vitro efficacy does not match with the in vivo
efficacy is not a rare phenomena. Perhaps the absorption, delivery,
distribution and metabolism are partly responsible for the
mentioned phenomena of 3aer.
duced into
procedure to give N-isoquinoline-3-carbonyl-
2
by the DCC/HOBt/N-methylmorpholine (NMM)
-amino acid benzy-
L
lesters (3aer, 53e87% yield). The mild condition and the acceptable
yields suggest that this three-step-route is suitable for preparing
these novel compounds.
2.4. Dose-dependence of the oral anti-tumor activity of 3g, n, q
The oral administration of 3g, n, q was observed at the doses of
2.2. In vitro anti-proliferation activity of 3aer
1, 10 and 100 mmol/kg to see the possible dose-dependent in vivo
anti-tumor response of S180 mice. The data are listed in Table 3, and
demonstrate that the tumor weight of the treated mice is
progressively increased with dose decrease. Therefore, oral 3g, n, q
dose-dependently inhibit the tumor growth of the treated S180
mice. This also suggests that 3aer should allow oral administration.
Following the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide (MTT) method, the in vitro anti-proliferation
assays were carried out on 96 microtiter plates, and the IC50 values
of 3aer inhibiting the growth of HL-60 and Hela cells were
R
O
CO2H
iii
CO2H
i
CO2H
ii
O-CH2C6H5
NH
NH2
NH
N
N
O
1
2
3a-r
Scheme 1. Synthetic route of N-isoquinoline-3-carbonyl-L-amino acid benzylesters. (i) HCHO and HCl; (ii) DMF and KMnO4; (iii) L-AA-OCH2C6H5, DCC and NMM; In 3a R ¼ CH3, 3b
R ¼ CH(CH3)2, 3c R ¼ CH(CH3)CH2CH3, 3d R ¼ indol-3-ylmethyl, 3e R ¼ CH2OH, 3f R ¼ 4-hydroxylbenzyl, 3g eNHCHRe ¼ cyclobutylamino, 3h R ¼ CH2CH2CONH2, 3i R ¼ imidazol-4-
ylmethylene, 3j R ¼ 4-aminobutyl-1-yl, 3k R ¼ benzyloxycarbonylmethyl, 3l R ¼ 2-benzyloxycarbonylethyl-1-yl, 3m R ¼ benzyl, 3n R ¼ CH(OH)CH3, 3o R ¼ CH2CH2SCH3, 3p R ¼ H,
3q R ¼ CH2CH(CH3)2, 3r R ¼ CH2CONH2.
Table 1
The IC50 values of 3aer inhibiting proliferation of HL-60 and Hela cells.
Compound
IC50 (X ꢂ SD nM, n ¼ 6)
Compound
IC50 (X ꢂ SD nM, n ¼ 6)
HL-60
Hela
HL-60
Hela
3a
3b
3c
3d
3e
3f
3g
3h
3i
84.84 ꢂ 5.81
78.94 ꢂ 6.64
96.35 ꢂ 5.31
66.12 ꢂ 9.72
70.39 ꢂ 9.16
99.94 ꢂ 7.91
53.42 ꢂ 9.44
44.50 ꢂ 5.92
87.56 ꢂ 6.16
68.24 ꢂ 7.64
154.46 ꢂ 12.62
118.29 ꢂ 5.20
185.42 ꢂ 12.34
396.46 ꢂ 19.47
134.65 ꢂ 13.12
507.94 ꢂ 15.41
87.74 ꢂ 13.84
180.67 ꢂ 12.42
263.43 ꢂ 15.24
195.67 ꢂ 10.38
3k
3l
73.96 ꢂ 4.91
83.62 ꢂ 5.72
91.79 ꢂ 9.52
62.25 ꢂ 5.64
44.88 ꢂ 6.93
57.35 ꢂ 7.63
72.76 ꢂ 6.72
86.94 ꢂ 6.81
948.52 ꢂ 12.4
411.30 ꢂ 15.31
213.61 ꢂ 16.52
151.00 ꢂ 10.81
98.69 ꢂ 9.74
3m
3n
3o
3p
3q
3r
2
308.75 ꢂ 15.56
195.43 ꢂ 10.31
110.55 ꢂ 8.94
216.31 ꢂ 14.33
1320.15 ꢂ 16.8
3j

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M. Zheng et al. / European Journal of Medicinal Chemistry 46 (2011) 1672e1681
Table 2
Effect of intraperitoneal 3aer on tumor growth of treated S180 mice.
Compounda
Tumor weight
% Inhibition
Compounda
Tumor weight
%Inhibition
3a
3b
3c
3d
3e
3f
3g
3h
3i
1.071 ꢂ 0.086b
1.266 ꢂ 0.094b
1.425 ꢂ 0.085b
1.137 ꢂ 0.105b
1.074 ꢂ 0.216b
1.297 ꢂ 0.103b
1.001 ꢂ 0.050b
1.588 ꢂ 0.102b
1.364 ꢂ 0.234b
1.493 ꢂ 0.078b
1.127 ꢂ 0.114b
40.6 ꢂ 4.3
29.7 ꢂ 2.3
20.9 ꢂ 3.1
36.9 ꢂ 2.6
40.4 ꢂ 3.8
28.0 ꢂ 3.0
44.5 ꢂ 5.2
11.9 ꢂ 2.4
24.3 ꢂ 3.1
40.6 ꢂ 4.3
37.5 ꢂ 4.1
3l
3m
3n
3o
3p
3q
3r
2
1.347 ꢂ 0.061b
1.260 ꢂ 0.204b
0.821 ꢂ 0.043c
1.088 ꢂ 0.164b
1.511 ꢂ 0.041b
0.953 ꢂ 0.126c
1.140 ꢂ 0.151b
1.595 ꢂ 0.300
0.814 ꢂ 0.066b
1.802 ꢂ 0.063
25.2 ꢂ 3.8
30.1 ꢂ 3.7
54.4 ꢂ 5.6
39.6 ꢂ 2.8
16.1 ꢂ 2.1
47.1 ꢂ 5.8
36.7 ꢂ 4.5
11.5 ꢂ 3.8
54.8 ꢂ 14.5
0
Cytarabine
NS
3j
3k
a
Tumor weight is represented by X ꢂ SD g; inhibition is represented by X ꢂ SD%; NS (normal saline) ¼ vehicle; n ¼ 12; Cytarabine, 2 and 3aer: dose ¼ 100
mmol/kg.
b
c
Compared to NS p < 0.01.
Compared to NS p < 0.01 and to cytarabine p > 0.05.
2.5. Neurotoxic and acute toxicity of 3g, n, q treated healthy mice
active orientation. To obtain a consistent alignment, isoquinoline
ring was selected as the template for superposing 3aer. The
method used for performing the alignment was the maximum
common subgraph (MCS) [47]. MCS looks at molecules as points
and lines, and uses the techniques out of graph theory to identify
the patterns. Then MCS finds the largest subset of atoms in iso-
quinoline ring that shared by 3aer. This subset was used for the
alignment. A rigid fit of atom pairings was performed to superim-
pose each structure onto the target model isoquinoline ring. Ster-
eoview of aligned 3aer is shown in Fig. 2. The alignment
stereoview explores that to superimpose onto isoquinoline ring the
amino acid residue of each structure has to take individual
conformation. As seen in Tables 2 and 3 this individual conforma-
tion of amino acid residue corresponds an individual anti-tumor
activity.
Neurotoxic and acute toxicity of the most potent 3g, n, q was
assayed by use of a general procedure on the healthy mice. It was
found that during 7-day observation the healthy mice orally
receiving 15, 90 and 540 mg/kg of 3g, n, q exhibited no any
neurotoxic behavior, such as tremor, twitch, jumping, tetanus, as
well as supination. On the 7th day the necropsy findings of the mice
gave also no apparent changes in their organs. These observations
suggest that 3g, n, q are low toxic agents. Besides, even up to
540 mg/kg of dose the treated healthy mice occurred no death. This
observation suggests that the LD50 value of 3g, n, q is more than
540 mg/kg. At the dose of 540 mg/kg the treated mice having 100%
survival and without neurotoxic behavior implies that 3g, n, q are
safe anti-tumor leads.
2.6.2. MFA based Cerius2 QSAR module of 3aeo
2.6. 3D QSAR analysis of 3aer
Molecular field analysis (MFA) was performed for 3aeo with the
QSAR module of Cerius2 [48]. A five-step-procedure consisted of
generating conformers, energy minimization, matching atoms and
To understand the dependence of the in vitro and in vivo anti-
tumor activity of 3aer upon their structure, the corresponding 3D
QSAR analysis was performed. Training set (3aeo)/test set (3per)
selections were done manually such that they populate the wide
range of anti-tumor activity in similar proportion. In the analysis,
the alignment, MFA based Cerius2 QSAR module of 3aer and the
electrostatic and steric environments of them within the grid with
3D points of the equation were involved.
2.6.1. Alignment of 3aer
For establishing the valid 3D-QSAR models, a proper alignment
procedure of 3aer was practiced using the target model align
strategy in the align module within Cerius2. Based on the
assumption that each structure of 3aer exhibits activity at the same
binding site of the receptor, they were aligned in a pharmacological
Table 3
Effect of different oral doses of 3g, n, q on tumor growth of treated S180 micea.
Compound
Tumor weight
mol/kg
1
m
10
m
mol/kg
100 mmol/kg
3g
3n
3q
NS
1.661 ꢂ 0.076b
1.315 ꢂ 0.078b
1.298 ꢂ 0.094b
1.879 ꢂ 0.127
1.243 ꢂ 0.181c
1.184 ꢂ 0.167c
1.011 ꢂ 0.085c
Cytarabine (100
0.814 ꢂ 0.066d
1.021 ꢂ 0.044d
0.802 ꢂ 0.043d
0.938 ꢂ 0.117d
m
mol/kg):
a
Tumor weight is represented by X ꢂ SDg, NS ¼ vehicle; n ¼ 12; cytarabine was
intraperitoneally administered.
b
Compared to NS p < 0.01.
Compared to NS p < 0.01, to cytarabine p > 0.05.
c
d
Intraperitoneal injection.
Fig. 2. Alignment stereoview of 3aer used for molecular field generation.

M. Zheng et al. / European Journal of Medicinal Chemistry 46 (2011) 1672e1681
1675
Table 4
Predict and test in vitro anti-proliferation activities of 3per.
Compound
IC50 (nM)
Predict value
Test value
Error
Error%
3p
3q
3r
60.80
69.70
82.96
57.35
72.76
86.94
ꢁ3.45
3.06
3.98
ꢁ6.0
4.2
4.6
was set for both electrostatic and steric fields. The total grid points
generated were 672. Though the spatial and structural descriptors
such as dipole moment, polarizability, radius of gyration, number of
rotatable bonds, molecular volume, principal moment of inertia,
AlogP98, numberof hydrogenbond donorsand acceptors, and molar
refractivity were also considered, only the highest variance holder
proton and methyl descriptors were used. Regression analysis was
carried out using the genetic partial least squares (G/PLS) method
consisting of 50,000 generations with a population size of 100. The
numberof components was set to5. Cross-validationwas performed
with the leave-one-out procedure. PLS analysis was scaled, with all
variables normalized to a variance of 1.0.
Fig. 3. Steric or electrostatic features of 3aer led to increasing or decreasing of anti-
proliferation of HL60 in vitro.
2.6.2.1. 3D QSAR equation of 3aeo in terms of inhibiting the plolif-
eration of HL60 in vitro. Based on the module, the regions where
variations in the steric or electrostatic features of 3aeo led to the
increase or decrease of their in vitro inhibition of proliferation of
HL60 were specified as Fig. 3. In the MFA model, the activities of
3aeo inhibiting the proliferation of HL60 in terms of the most
relevant descriptors including methyl and hydroxyl anion is
expressed with Eq. (1).
IC50ðnMÞ ¼ 48:06 þ 0:68ðCH3=625Þ þ 0:84ðCH3=835Þ
þ 0:53ðCH3=944Þ ꢁ 0:63ðCH3=484Þ
ꢀ
ꢁ
þ 0:28 HOꢁ=596
(1)
The correlation of the activities tested on the in vitro plolifera-
tion model of HL60 and the activities calculated with Eq. (1) is
explained with Fig. 4. In Eq. (1) the data points (n), correlation
coefficient (r) and square correlation coefficient (r2) were 15, 0.989
and 0.978, respectively. These parameters indicate that Eq. (1) is
able to predict the in vitro activity for 3aeo.
Fig. 4. Graph of tested in vitro anti-proliferation activity versus predicted in vitro anti-
proliferation activity of 3aer.
Eq. (1) contains 4 terms from methyl descriptor and 1 term from
hydroxyl anion descriptor. The terms of 0.68 (CH3/625), 0.84 (CH3/
835) and 0.53 (CH3/944) have positive coefficients, which means
that at these positions small groups will increase the activity, while
the term of 0.63 (CH3/484) has negative coefficient, which means
that at this position large group will increase the activity. The term
of 0.28 (HOꢁ/596) has positive coefficient, which means that at this
position hydrogen bond forming group will decrease the activity.
aligning molecules, setting preferences, and regression analysis was
automatically practiced in MFA. Molecular electrostatic and steric
fields were created by use of proton, methyl and hydroxyl anion as
probes, respectively. These fields were sampled at each point of
a regularly spaced grid of 1 Å. An energy cutoff of ꢂ30.0 kcal/mol
Fig. 5. Electrostatic and environments of 3c and 3h within the grid with 3D points of Eq. (1).

1676
M. Zheng et al. / European Journal of Medicinal Chemistry 46 (2011) 1672e1681
Table 5
Predict and test in vivo anti-tumor activities of 3per.
Compound
Tumor weight (g)
Predict value
Test value
Error
Error%
3p
3q
3r
1.446
1.006
1.130
1.511
0.953
1.140
ꢁ0.065
0.053
ꢁ0.01
ꢁ4.3
5.6
ꢁ0.87
near the region of CH3/835, thus it totally has high in vitro anti-
proliferation activity.
2.6.2.2. Predicting the in vitro anti-proliferation activity of 3per with
Eq. (1). The predict power of Eq. (1) was demonstrated by
comparing the calculated and tested in vitro anti-proliferation
activity of 3per (Table 4). The correlations of the predict and the
test values are also shown in Fig. 4. The results indicate that Eq. (1)
rationally gives in vitro anti-proliferation activity for 3per and the
errors range from ꢁ3.45 to 3.98 nM. The calculated activity is so
approximate to experimental activity means that Eq. (1) is practical
to accurately predict the in vitro anti-proliferation activity of iso-
Fig. 6. Steric or electrostatic features of 3aer led to increasing or decreasing of anti-
tumor activity in vivo.
quinoline-3-carboxyl-L-amino acid benzylesters.
2.6.2.3. 3D QSAR equation of 3aeo inhibiting tumor growth of
treated S180 mice. Based on the module, the regions where varia-
tions in the steric or electrostatic features of 3aer lead to the
inhibition or enhancement of tumor growth of the treated S180
mice were specified as in Fig. 6. In the MFA model, the in vivo anti-
tumor activity of 3aeo in terms of the most relevant descriptors
including methyl and hydroxyl anion is expressed by Eq. (2).
ꢀ ꢁ
Tumorweight g ¼0:433þ0:0022ðCH3=715Þþ0:010ðCH3=838Þ
ꢀ
ꢁ
ꢁ0:020ðCH3=465Þþ0:0058 HOꢁ=833
ꢀ
ꢁ
þ0:0049 HOꢁ=838
ð2Þ
The correlation of the activities tested on the in vivo anti-tumor
model and calculated using Eq. (2) is explained with Fig. 7. In
equation 2 the data points (n), correlation coefficient (r) and square
correlation coefficient (r2) were 15, 0.987 and 0.975, respectively.
The parameters indicated that Eq. (2) is able to predict the in vivo
activity for 3aeo.
Fig. 7. Graph of tested versus predicted in vivo anti-tumor activities of 3aer.
Eq. (2) contains 3 terms from methyl descriptor and 2 terms
from hydroxyl anion descriptor. The terms of 0.0022 (CH3/715) and
0.010 (CH3/838) have positive coefficients, which means that at
these positions small groups will increase the activity, while term
of 0.020 (CH3/465) has negative coefficient, which means that at
this position large group will increase the activity. The terms of
0.0058 (HOꢁ/833) and 0.0049 (HOꢁ/838) have positive coefficients,
Fig. 5 gives 3c, h as diagrammatic examples of Eq. (1) Compound
3c has a small group near the region of CH3/484 and a hydrogen
bond forming group near the region of HOꢁ/596, thus it has low in
vitro anti-proliferation activity. Compound 3h has small group near
the regions of CH3/625 and 0.53 CH3/944, as well as a large group
Fig. 8. Electrostatic and environments of 3c and 3n within the grid with 3D points of Eq. (2).

M. Zheng et al. / European Journal of Medicinal Chemistry 46 (2011) 1672e1681
1677
Table 6
Information) defines a concentration-dependent decrease of fluo-
rescence intensities (fluorescence quenching) of 3n suggests that
the action mechanism should be DNA intercalation and ensures
that 3aer should be DNA intercalators [49]. According to this
comment the topoisomerase I induced cleavage of dimeric pRR322
DNA assays were performed for compounds 2 and 3n, o, q, the
results were described with the electrophoresis of the reaction
mixtures were analyzed using a 0.8% agarose gel in TRE buffer
(Fig. 2 of Supporting Information). The electrophoresis indicates
that compounds 2 and 3n, o, q are the inhibitors of topoisomeruse I.
Apparent permeability coefficients of 2 and 3n, q.
Compound
Papp ꢀ 106(cm/s)a
A / B
B / A
A / B/B / A
2
3n
3q
9.47
18.61
17.22
9.28
7.05
7.12
1.02
2.64
2.42
a
2 and 3n, q were dissolved in HBSS (final concentration: 4 mM), the standard
deviations were generally less than 9.2% (n ¼ 4); A / B: from apical side to baso-
lateral side; B / A: from basolateral side to apical side.
4. Experimental section
which means that at these positions hydrogen bond forming
groups will decrease the activity.
4.1. General method
Fig. 8 gives 3c,n as diagrammatic examples of equation 2.
Compound 3c has hydrogen bond forming groups near the regions
of HOꢁ/833 and HOꢁ/838 and therefore possesses low in vivo anti-
tumor activity. Compound 3n has a small group near the region of
CH3/838 and therefore possesses high in vivo anti-tumor activity.
All the reactions were carried out under nitrogen (1 bar). 1H
(300 and 500 M Hz) and 13C (75 and 125 M Hz) NMR spectra were
recorded on Bruker AMX-300 and AMX-500 spectrometers for
solution DMSO-d6, or CDCl3 with tetramethylsilane as internal
standard. IR spectra were recorded with a PerkineElmer 983
instrument. FAB/MS was determined on VG-ZAB-MS, and TOF-MS
was recorded on MDS SCIEX QSTAR. Melting points were measured
on a XT5 hot stage microscope (Beijing key electro-optic factory).
2.6.2.4. Predicting in vivo anti-tumor activity of 3per with Eq.
(2). The predict power of Eq. (2) was demonstrated by comparing
the calculated and the tested in vivo anti-tumor activities of 3per
(Table 5). The correlation of the predict value and the test value is
also shown in Fig. 7. The results indicate that Eq. (2) rationally gives
the in vivo anti-tumor activity for 3per and the errors range
from ꢁ0.01 to 0.053 g. The calculated activity is so approximate to
the experimental activity means that Eq. (2) is practical to accu-
rately predict the in vitro anti-tumor activity of isoquinoline-3-
All L-amino acids were purchased from China Biochemical Corp. TLC
was made with Qingdao silica gel GF254. Chromatography was
performed with Qingdao silica gel H60 or Sephadex-LH20. The
purities of the intermediates were measured on TLC (Merck silica
gel plates of type 60 F254, 0.25 mm layer thickness, three systems of
mixed solvents) and gave a point of single component, while the
purities of the products were measured on HPLC (Waters, C18
column 4.6 ꢀ 150 mm, three elution systems of mixed solvents) and
more than 96%. All solvents were distilled and dried before use
according to literature procedures. Optical rotations were deter-
mined with a Jasco P-1020 Polarimeter at 20 ꢃC. The statistical
analysis of all the biological data was carried out by use of ANOVA
test, p < 0.05 is considered significant.
carbonyl-L-amino acid benzylesters.
2.7. In vitro membrane permeation tests of 2 and 3n, q
To know the contribution of this modification to the
enhancement of the permeability the membrane permeability of
isoquinoline-3-carboxylic acid and the most potent isoquinoline-
3-carbonyl-
carbonyl- -leucine benzylester (3q) were measured on Caco-2 cell
monolayers, and the data are summarized in Table 6. The data
indicate that modifying isoquinoline-3-carboxylic acid with -Thr-
OBzl and -Leu-OBzl results in a significant increase of the
L-threonine benzylester (3n) and isoquinoline-3-
L
4.2. Preparing compounds
L
4.2.1. (3S)-1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid (1)
L
To the suspension of 5.0 g (0.03 mmol) of L-Phe in 50 ml of
permeability. Though the bioavailability of a drug is influenced by
a series of factors including dissolution, absorption, pre-systemic
and systemic metabolism, and elimination, the intestinal mucosa
is a significant barrier to ip delivery of drugs into the systemic
circulation. Caco-2 cells possess many structural and functional
similarities to human enterocytes. After converting isoquinoline-
3-carboxylic acid into its deceptive analogue 3n, q the perme-
ability gains 2.5-fold increases.
chloroform and 27 ml of formaldehyde 45 ml of concentrated
hydrochloric acid was added drop-wise. The reaction mixture was
stirred at 80e90 ꢃC for 10 h, and TLC (CHCl3/CH3OH, 10:1) indicates
the complete disaprearance of L-Phe. The reaction mixture was
cooled to room temperature and the formed precipitates were
collected byfiltration. The collected solids weresuccessively washed
with water (30 ml ꢀ 3) and acetone (30 ml ꢀ 3) to give 4.5 g (83.9%)
of the title compound as a colorless powder. Mp 302e303 ꢃC;
20
[a
]
¼ ꢁ68 (c ¼ 1.0, H2O); ESI-MS (m/e) 178 [M þ H]þ; 1H NMR
D
3. Conclusion
(300 MHz, CDCl3)
d
/ppm ¼ 11.0 (s, 1H), 7.25 (m, J ¼ 6.4 Hz, 2H), 7.02
(d, J ¼ 6.5 Hz, 1H), 6.98 (t, J ¼ 6.6 Hz, 1H), 3.80 (m, 3H), 3.03 (d,
In conclusion, based on the fact that the in vitro anti-tumoral
activity of isoquinoline-3-carboxylic acid is at least 80-fold higher
J ¼ 7.5 Hz, 1H), 2.78 (d, J ¼ 8.4 Hz, 1H), 2.0(s, 1H); 13C NMR (75 MHz,
CDCl3)
d/ppm ¼ 174.9, 136.2, 134.2, 127.2, 126.0, 57.6, 47.4, 29.4.
than that of
quinoline-3-carboxylic acid is a desirable lead compound. The
modification of isoquinoline-3-carboxylic acid with -amino acid
b-carboline-3-carboxylic acid it is national that iso-
4.2.2. Isoquinoline-3-carboxylic acid (2)
L
At 0 ꢃC to a solution of 10.0 g (0.046 mmol) of (3S)-1,2,3,4-tet-
rahydroisoquinoline-3-carboxylic acid in 200 ml of N,N-dime-
thylformamide 5.0 g (0.032 mmol) of KMnO4 was added, which
took 30 min. The reaction mixture was stirred at room temperature
for 100 h, and TLC (CCl3:CH3OH ¼ 5:1) indicated the complete
disappearance of (3S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic
acid. The reaction mixture was evaporated under vacuum. The
residue was washed with distilled water repeatedly to provide 8.1 g
(83%) of the title compound as a yellow powder. ESI-MS (m/e) 174
benzylesters resulted in a class of novel isoquinoline-3-carbon-
ylamino acid benzylesters. The modification resulted in 2.5-fold
increases of the membrane permeability, 2.6-fold to 21.3-fold
increases of the in vitro anti-proliferation activity and 1.0-fold to
4.7-fold increases of the in vivo anti-tumor activity. In addition, on
S180 mouse model even if the oral dose was lowed to 1 mmol/kg 3g,
n, q still effectively inhibited the tumor growth of the treated
S180 mice. The preliminary fluorescence study (see Supporting

1678
M. Zheng et al. / European Journal of Medicinal Chemistry 46 (2011) 1672e1681
[M þ H]þ; 1H NMR (300 MHz, DMSO)
d
/ppm ¼ 10.81 (s,1 H), 9.52 (s,
1379, 1087, 1050, 881, 800, 620 cmꢁ1; 1H NMR (300 MHz, DMSO)
d/
1 H), 8.43 (s, 1 H), 7.52 (m, 4 H); 13C NMR (75 MHz, DMSO)
d/
ppm ¼ 10.11 (s, 1 H), 9.44 (s, 1 H), 8.58 (s, 1 H), 8.24 (q, J ¼ 8.4 Hz, 2
H), 8.01 (d, J ¼ 8.6 Hz, 1 H), 7.86 (m, 2 H), 7.19 (m, 9 H), 6.85 (d,
J ¼ 8.6 Hz, 1 H), 5.34 (s, 2 H), 4.81 (q, J ¼ 8.4 Hz, 1 H), 3.02 (m, 2 H);
ppm ¼ 164.9, 152.3, 143.5, 136.2, 131.3, 129.7, 127.2, 125.8.
4.3. General procedure for preparing 3aer
13C NMR (75 MHz, DMSO)
138.6, 132.1, 131.2, 129.6, 129.0, 128.5, 124.8, 122.8, 122.3, 120.1,
119.2, 111.3, 110.5, 68.7, 58.0, 28.9; Anal. Calcd for C28H23N3O3: C,
74.82; H, 5.16; N, 9.35. Found C, 74.60; H, 5.31; N, 9.12.
d
/ppm ¼ 171.9, 165.1, 152.1, 150.8, 143.6,
At 0 ꢃC and with stirring to the solution of 865 mg (5.0 mmol) of
isoquinoline-3-carboxylic acid in 10 ml of anhydrous THF 675 mg
(5.0 mmol) of HOBt was added to form reaction mixture A. The
solution of 5.5 mmol of
L-amino acid benzylester in 5 ml of anhydrous
4.3.5. Benzyl (S)-2-hydroxymethyl-2-(isoquinoline-3-carboxamido)
THF was adjusted pH 9 with triethylamine and stirred for 30 min to
form mixture B. At 0 ꢃC the mixtures A and B were mixed and then
1339 mg (6.5 mmol) of DCC was added. The reaction mixture was
stirred at 0 ꢃC for 2 h, at room temperature for12 h and TLC (ethyl
acetate/petroleum ether, 1:2) indicated the complete disappearance
of isoquinoline-3-carboxylic acid. The formed precipitates of DCU
were removed by filtration and the filtrate was evaporated under
vacumm. The residue was dissolved in 50 ml of ethyl acetate and the
formed solution was washed successively with saturated aqueous
solution of NaHCO3 (30 ml ꢀ 3), 5% aqueous solution of KHSO4
(30 ml ꢀ 3) and saturated aqueous solution of NaCl (30 ml ꢀ 3) and
dried over anhydrous Na2SO4. After filtration the filtrate was evapo-
rated under vacumm and the residure was purified on silica gel
chromatography (CHCl3:MeOH, 20:1) to give the title compounds.
acetate (3e)
Yield: 84%. Colorless powders. ESI-MS (m/e) 441 [M þ H]þ;
D
[a
]
¼ ꢁ7.1 (c ¼ 1.0, CH3OH); IR (KBr): 3413, 2969, 1734, 1648,
20
1497, 1457, 1440, 1198, 1111 cmꢁ1 1H NMR (300 MHz, DMSO)
; d/
ppm ¼ 9.44 (s, 1 H), 8.75 (d, J ¼ 8.6 Hz, 1 H), 8.58 (s, 1 H), 8.24 (q,
J ¼ 8.4 Hz, 2 H), 7.86 (m, 2 H), 7.32 (m, 10 H), 5.32 (s, 2 H), 4.74 (m, 1
H), 4.61 (s, 2 H), 4.01 (m, 2 H); 13C NMR (75 MHz, DMSO)
d/
ppm ¼ 170.6, 164.6, 152.2, 143.4, 139.0, 137.6, 132.1, 129.6, 128.5,
127.2, 124.8, 79.2, 72.4, 66.7, 53.0; Anal. Calcd for C27H24N2O4: C,
73.62; H, 5.49; N, 6.36. Found C, 73.40; H, 5.62; N, 6.11.
4.3.6. Benzyl (S)-2-(4-hydroxylbenzyl)-2-(isoquinoline-3-
carboxamido)acetate (3f)
Yield: 56%. Colorless powders. ESI-MS (m/e) 427 [M þ H]þ;
D
[a
]
¼ ꢁ3.2 (c ¼ 1.0, CH3OH); IR (KBr): 3420, 2969, 1734, 1648,
20
4.3.1. Benzyl (S)-2-methyl-2-(isoquinoline-3-carboxamido)acetate
1600, 1497, 1450, 1440, 1198, 1110 cmꢁ1; 1H NMR (300 MHz, DMSO)
(3a)
d
/ppm ¼ 9.44 (s, 1 H), 8.75 (d, J ¼ 8.6 Hz, 1 H), 8.58 (s, 1 H), 8.24 (q,
Yield: 87%. Colorless powders. ESI-MS (m/e) 335 [M þ H]þ;
J ¼ 8.4 Hz, 2 H), 7.86 (m, 2 H), 7.22 (m, 5 H), 6.96 (d, J ¼ 8.8 Hz, 2 H),
6.69 (d, J ¼ 8.8 Hz, 2 H), 5.17 (s, 2 H), 4.74 (m, 1 H), 3.31 (dd,
J ¼ 3.6 Hz, 12.1 Hz, 1 H), 3.05 (dd, J ¼ 3.6 Hz, 12.1 Hz, 1 H); 13C NMR
D
[
a]
¼ ꢁ6.6 (c ¼ 1.0, CH3OH); IR (KBr): 3312, 2972, 2960, 2883,
20
2870, 1710, 1690, 1450, 1380 cmꢁ1 1H NMR (300 MHz, DMSO)
; d/
ppm ¼ 9.41 (s,1 H), 9.16 (s,1 H), 8.59 (s,1 H), 8.24 (q, J ¼ 8.4 Hz, 2 H),
(75 MHz, DMSO)
d
/ppm ¼ 170.6, 164.6, 155.6, 152.2, 151.7, 143.4,
7.85 (m, 2 H), 7.35(m, 5 H), 5.18 (s, 2 H), 4.70 (m, 1 H), 1.51 (d,
137.6, 132.1, 131.5, 128.5, 124.8, 79.2, 72.4, 66.7, 57.0, 16.7; Anal.
Calcd for C26H22N2O4: C, 73.23; H, 5.20; N, 6.57. Found C, 73.00; H,
5.34; N, 6.23.
J ¼ 8.6 Hz, 3 H); 13C NMR (75 MHz, DMSO)
/ppm ¼ 172.7, 164.9,
d
152.8, 151.0, 142.9, 138.2, 131.6, 129.6, 129.1, 127.6, 127.3, 124.8, 66.7,
48.6, 17.6. Anal. Calcd for C20H18N2O3: C, 71.84; H, 5.43; N, 8.38.
Found C, 71.67; H, 5.54; N, 8.15.
4.3.7. Benzyl (S)-2-(1-isoquinoline-3-carbonylpyrrolidine-2-yl)
acetate (3g)
4.3.2. Benzyl (S)-2-(2-propyl)-2-(isoquinoline-3-carboxamido)
acetate (3b)
Yield: 76%. Colorless powders. ESI-MS (m/e) 361 [M þ H]þ;
D
[a
]
¼ ꢁ2.0 (c ¼ 1.0, CH3OH); IR (KBr): 3411, 2969, 1734, 1650,
20
Yield: 87%. Colorless powders. ESI-MS (m/e) 363 [M þ H]þ;
1497, 1457, 1375, 1198, 1111, 750, 599 cmꢁ1
DMSO)
;
1H NMR (300 MHz,
/ppm ¼ 9.44 (s, 1 H), 8.58 (s, 1 H), 8.24 (q, J ¼ 8.4 Hz, 2 H),
D
[
a
]
¼ ꢁ4.9 (c ¼ 1.0, CH3OH); IR (KBr): 3312, 2972, 2961, 2890,
d
20
2869, 1710, 1690, 1385, 1375, 599 cmꢁ1; 1H NMR (300 MHz, DMSO)
/ppm ¼ 9.42 (s, 1 H), 8.60 (s, 1 H), 8.24 (q, J ¼ 8.4 Hz, 2 H), 7.86 (m, 2
7.86 (m, 2 H), 7.32 (m, 5 H), 5.34 (s, 2 H), 4.18 (t, J ¼ 8.5 Hz, 1 H), 3.35
d
(t, J ¼ 8.5 Hz, 2 H), 1.96 (q, J ¼ 8.4 Hz, 2 H), 1.60 (m, 2 H); 13C NMR
H), 7.36 (m, 5 H), 5.21 (q, J ¼ 8.4 Hz, 2 H), 4.57 (t, J ¼ 8.5 Hz,1 H), 2.30
(75 MHz, DMSO)
d/ppm ¼ 171.9, 165.1, 152.1, 150.8, 143.6, 138.6,
(m, 1 H), 0.95 (d, J ¼ 8.6 Hz, 6 H); 13C NMR (75 MHz, DMSO)
d/
132.1, 129.6, 129.1, 128.5, 127.6, 124.8, 68.7, 58.0, 46.1, 28.9, 22.1;
Anal. Calcd for C22H20N2O3: C, 73.32; H, 5.59; N, 7.77. Found C,
73.11; H, 5.75; N, 7.54.
ppm ¼ 171.6, 164.5, 152.3, 151.3, 143.0, 136.0, 132.1, 128.6, 124.5,
66.7, 57.0, 30.8, 25.4, 19.0. Anal. Calcd for C22H22N2O3: C, 72.91; H,
6.12; N, 7.73. Found C, 72.70; H, 6.25; N, 7.50.
4.3.8. Benzyl (S)-2-(2-aminocarbonylethyl-1-yl)-2-(isoquinoline-3-
4.3.3. Benzyl (S)-2-(2-butyl)-2-(isoquinoline-3-carboxamido)
carboxamido)-acetate (3h)
acetate (3c)
Yield: 53%. Colorless powders. ESI-MS (m/e) 392 [M þ H]þ;
D
Yield: 75%. Colorless powders. ESI-MS (m/e) 377 [M þ H]þ;
[a
]
¼ 3.33 (c ¼ 1.0, CH3OH); IR (KBr): 3414, 2970, 1734, 1648,
20
D
[
a]
¼ ꢁ4.2 (c ¼ 1.0, CH3OH); IR (KBr): 3312, 2972, 2960, 2890,
1498, 1457,1375, 1198, 750, 599 cmꢁ1; 1H NMR (300 MHz, DMSO)
d/
20
2871,1710,1690,1450,1380, 600 cmꢁ1; 1HNMR (300 MHz, DMSO)
d
/
ppm ¼ 9.44 (s, 1 H), 8.75 (d, J ¼ 8.6 Hz, 1 H), 8.58 (s, 1 H), 8.24 (q,
J ¼ 8.4 Hz, 2 H), 7.86(m, 2 H), 7.32 (m, 5 H), 6.0 (s, 2 H), 5.34 (s, 2 H),
4.45 (q, J ¼ 8.4 Hz, 1 H), 2.98 (q, J ¼ 8.4 Hz, 2 H), 2.16 (q, J ¼ 8.4 Hz, 2
ppm ¼ 9.42 (s,1 H), 8.80 (d,1 H), 8.59 (s,1 H), 8.24 (q, J ¼ 8.4 Hz, 2 H),
7.86 (m, 2 H), 7.36 (m, 5 H), 5.21 (s, 2 H), 4.62 (t, J ¼ 8.5 Hz, 1 H), 2.51
(m, 1 H), 1.30 (m, 2 H), 0.96 (m, 6 H); 13C NMR (75 MHz, DMSO)
d/
H); 13C NMR (75 MHz, DMSO)
d/ppm ¼ 172.6, 171.8, 165.0, 152.1,
ppm ¼ 171.6, 164.4, 152.3, 143.3, 136.0, 132.1, 131.2, 129.5, 128.6,
127.6, 127.2, 120.5, 66.7, 57.0, 37.2, 25.4, 16.0, 11.6; Anal. Calcd for
C23H24N2O3: C, 73.38; H, 6.43; N, 7.44. Found C, 73.15; H, 6.57; N, 7.22.
150.8, 143.6, 138.6, 132.1, 128.5, 127.6, 127.2, 124.8, 68.7, 52.0, 33.1,
28.3; Anal. Calcd for C22H21N3O4: C, 67.51; H, 5.41; N,10.74. Found C,
67.31; H, 5.55; N, 10.51.
4.3.4. Benzyl (S)-2-(1H-indol-3-ylmethyl)-2-(isoquinoline-3-
4.3.9. Benzyl (S)-2-(imidazol-4-ylmethyl)-2-(isoquinoline-3-
carboxamido)acetate (3d)
carboxamido)acetate (3i)
Yield: 59%. Colorless powders. ESI-MS (m/e) 450 [M þ H]þ;
Yield: 55%. Colorless powders. ESI-MS (m/e) 401 [M þ H]þ;
D
D
[
a]
¼ ꢁ3.1 (c ¼ 1.0, CH3OH); IR (KBr): 3410, 3312, 2972, 2883,
[a
]
¼ ꢁ4.7 (c ¼ 1.0, CH3OH); IR (KBr): 3420, 2969, 1734, 1648,
20
20

M. Zheng et al. / European Journal of Medicinal Chemistry 46 (2011) 1672e1681
1679
1497, 1457, 1375, 1198, 1200, 750 cmꢁ1; 1H NMR (300 MHz, DMSO)
J ¼ 8.4 Hz, 2 H), 7.86 (m, 2 H), 7.32 (m, 10 H), 5.17 (s, 2 H), 4.74 (m, 1
H), 4.61 (s, 2 H), 4.27 (m, 1 H), 1.26 (d, J ¼ 8.6 Hz, 3 H); 13C NMR
d
/ppm ¼ 13.12 (s, 1 H), 9.44 (s, 1 H), 8.58(s, 1 H), 8.24 (q, J ¼ 8.4 Hz, 2
H), 8.02 (d, J ¼ 8.6 Hz, 1 H), 7.86 (m, 2 H), 7.44 (s, 1 H), 7.19 (m, 5 H),
6.82 (d, J ¼ 8.6 Hz,1 H), 5.34 (s, 2 H), 4.81 (q, J ¼ 8.4 Hz,1 H), 3.12 (m,
(75 MHz, DMSO)
d
/ppm ¼ 170.6, 164.6, 152.2, 150.8, 143.4, 137.6,
132.1, 131.0, 129.5, 128.5, 127.9, 127.2, 124.8, 79.2, 72.4, 66.7, 57.0,
16.7; Anal. Calcd for C28H26N2O4: C, 73.99; H, 5.77; N, 6.16. Found C,
73.76; H, 5.94; N, 5.93.
2 H); 13C NMR (75 MHz, DMSO)
d
/ppm ¼ 171.9, 165.1, 152.1, 150.8,
143.6, 138.6, 135.8, 132.1, 129.6, 129.1, 128.5, 127.2, 124.8, 110.5, 68.7,
58.0, 28.9; Anal. Calcd for C23H20N4O3: C, 68.99; H, 5.03; N, 13.99.
Found C, 68.76; H, 5.18; N, 13.73.
4.3.15. Benzyl (S)-2-(2-methylmercaptoethyl-1-yl)-2-(isoquinoline-
3-carboxamido)-acetate (3o)
4.3.10. Benzyl (S)-2-(4-aminobutyl-1-yl)-2-(isoquinoline-3-
Yield: 83%. Colorless powders. ESI-MS (m/e) 395 [M þ H]þ;
D
carboxamido)acetate (3j)
[
a
]
¼ ꢁ1.3 (c ¼ 1.0, CH3OH); IR (KBr): 3410, 2969, 1734, 1648,
20
Yield: 90%. Colorless powders. ESI-MS (m/e) 392 [MþH]þ;
1497, 1457, 1380, 1198, 1113, 750 cmꢁ1 1H NMR (300 MHz, DMSO)
;
D
[
a]
¼ 3.6 (c ¼ 1.0, CH3OH); IR (KBr): 3411, 2969, 1735, 1651, 1496,
d
/ppm ¼ 9.44 (s, 1 H), 8.75 (d, J ¼ 8.6 Hz, 1 H), 8.58 (s, 1 H), 8.24 (q,
20
1458, 1376, 1198, 725, 600 cmꢁ1
;
1H NMR (300 MHz, DMSO)
d/
J ¼ 8.4 Hz, 2 H), 7.86 (m, 2 H), 7.32 (m, 5 H), 5.34 (s, 2 H), 4.45 (q,
J ¼ 8.4 Hz, 1 H), 2.48 (t, J ¼ 8.5 Hz, 2 H), 2.26 (q, J ¼ 8.4 Hz, 2 H), 2.10
ppm ¼ 9.44 (s, 1 H), 8.58 (s, 1 H), 8.24 (d, J ¼ 8.6 Hz, 2 H), 8.01 (d,
J ¼ 8.6 Hz, 1 H), 7.86 (m, 2 H), 7.19 (m, 5 H), 5.34 (s, 2 H), 4.41 (q,
J ¼ 8.4 Hz, 1 H), 2.62 (m, 2 H), 2.01 (t, J ¼ 8.5 Hz, 2 H), 1.90 (q,
J ¼ 8.4 Hz, 2 H), 1.55 (m, 2 H), 1.29 (m, 2 H); 13C NMR (75 MHz,
(s, 3 H); 13C NMR (75 MHz, DMSO)
143.6, 138.6, 132.1, 129.6, 129.0, 128.5, 127.7, 127.1, 124.8, 68.7, 52.0,
31.1, 30.3, 15.0; Anal. Calcd for C22H22N2O3S: C, 66.98; H, 5.62; N,
7.10. Found C, 66.77; H, 5.75; N, 6.88.
d
/ppm ¼ 171.9, 165.1,152.1, 150.8,
DMSO)
d
/ppm ¼ 171.9, 165.1, 152.1, 150.8, 143.6, 138.6, 132.1, 129.6,
128.5, 127.2, 127.0, 124.8, 110.5, 68.7, 53.0, 42.3, 32.5, 31.1, 21.1; Anal.
Calcd for C23H25N3O3: C, 70.57; H, 6.44; N, 10.73. Found C, 70.34; H,
6.61; N, 10.50.
4.3.16. Benzyl 2-(isoquinoline-3-carboxamido)acetate (3p)
Yield: 79%. Colorless powders. ESI-MS (m/e) 321 [M þ H]þ;
D
[a
]
¼ 5.6 (c ¼ 1.0, CH3OH); IR (KBr): 3312, 2972, 2961, 2883,
20
4.3.11. Benzyl (S)-2-benzyloxycarbonylmethyl-2-(isoquinoline-3-
2869, 1710, 1690, 1440 cmꢁ1 1H NMR (300 MHz, DMSO)
; d/
carboxamido)acetate (3k)
ppm ¼ 9.44 (s, 1 H), 8.68 (s, 1 H), 8.24 (q, J ¼ 8.4 Hz, 2 H), 7.87(m, 2
Yield: 77%. Colorless powders. ESI-MS (m/e) 483 [M þ H]þ;
H), 7.43 (m, 5 H), 7.31(s, 1 H), 5.44 (s, 2 H), 4.41 (d, J ¼ 8.6 Hz, 2 H);
[a]
¼ 22.1 (c ¼ 1.0, CH3OH); IR (KBr): 3411, 2969,1734, 1648,1497,
13C NMR (75 MHz, DMSO)
137.2, 130.6, 129.5, 129.1, 127.2, 124.9, 67.7, 41.6. Anal. Calcd for
C19H16N2O3: C, 71.24; H, 5.03; N, 8.74. Found C, 71.05; H, 5.17; N,
8.51.
d
/ppm ¼ 169.7, 161.9, 151.8, 151.0, 141.9,
D
20
1457, 1390, 1198, 750, 599 cmꢁ1
;
1H NMR (300 MHz, DMSO)
d/
ppm ¼ 9.44 (s, 1 H), 8.75 (d, J ¼ 8.6 Hz, 1 H), 8.58 (s, 1 H), 8.24 (q,
J ¼ 8.4 Hz, 2 H), 7.86 (m, 2 H), 7.32 (m, 10 H), 5.17 (m, 4 H), 4.45 (q,
J ¼ 8.4 Hz, 1 H), 2.51 (m, 4 H); 13C NMR (75 MHz, DMSO)
d/
ppm ¼ 172.6,171.8,165.0,152.1,143.6,138.6,132.1,129.5,128.5,127.4,
126.9, 124.8, 79.2, 72.4, 68.7, 52.0, 28.1, 26.7; Anal. Calcd for
C29H26N2O5: C, 72.18; H, 5.43; N, 5.81. Found C, 72.00; H, 5.55; N, 5.60.
4.3.17. Benzyl (S)-2-(2-methylpropyl-1-yl)-2-(isoquinoline-3-
carboxamido)acetate (3q)
Yield: 81%. Colorless powders. ESI-MS (m/e) 377 [M þ H]þ;
D
[
a
]
¼ ꢁ5.1 (c ¼ 1.0, CH3OH); IR (KBr): 3312, 2972, 2961, 2890,
20
4.3.12. Benzyl (S)-2-(2-benzyloxycarbonylethyl-1-yl)-2-
2869, 1710, 1690, 1385, 1375, 599 cmꢁ1; 1H NMR (300 MHz, DMSO)
/ppm ¼ 8.54 (s, 1 H), 8.08 (s, 1 H), 8.24 (q, J ¼ 8.4 Hz, 2 H), 7.87 (m, 2
(isoquinoline-3-carboxamido)-acetate (3l)
d
Yield: 83%. Colorless powders. ESI-MS (m/e) 469 [M þ H]þ;
H), 7.36 (m, 5 H), 7.31 (s, 1 H), 5.15 (s, 2 H), 4.42 (t, J ¼ 8.5 Hz, 1 H),
D
[
a]
¼ 13.1 (c ¼ 1.0, CH3OH); IR (KBr): 3413, 2969,1734,1648,1497,
1.58 (m, 3 H), 0.86 (d, J ¼ 8.6 Hz, 6 H); 13C NMR (75 MHz, DMSO)
d/
20
1457, 1375, 1198, 1111, 750, 599 cmꢁ1; 1H NMR (300 MHz, DMSO)
d/
ppm ¼ 172.4, 161.5, 152.8, 151.1, 142.9, 136.4, 128.6, 131.5, 129.6,
129.0, 127.6, 127.0, 124.9, 68.5, 48.7, 40.7, 23.8, 22.8; Anal. Calcd for
C23H24N2O3: C, 73.38; H, 6.43; N, 7.44. Found C, 73.16; H, 6.57; N,
7.20.
ppm ¼ 9.44 (s, 1 H), 8.75 (d, J ¼ 8.6 Hz, 1 H), 8.58 (s, 1 H), 8.24 (q,
J ¼ 8.4 Hz, 2 H), 7.86 (m, 2 H), 7.32 (m, 10 H), 5.17 (m, 4 H), 4.75 (q,
J ¼ 8.4 Hz, 1 H), 2.51 (m, 2 H); 13C NMR (75 MHz, DMSO)
d/
ppm ¼ 172.6,171.8,165.0,152.1,143.6,138.6,132.1,129.4,128.5,127.3,
127.0,124.8, 79.2, 72.4, 66.7, 47.0, 36.7; Anal. Calcd for C28H24N2O5: C,
71.78; H, 5.16; N, 5.98. Found C, 71.56; H, 5.31; N, 5.74.
4.3.18. Benzyl (S)-2-aminocarbonylmethyl-2-(isoquinoline-3-
carboxamido)acetate (3r)
Yield: 54%. Colorless powders. ESI-MS (m/e) 378 [M þ H]þ;
D
4.3.13. Benzyl (S)-2-benzyl-2-(isoquinoline-3-carboxamido)acetate
[a]
¼ 6.3 (c ¼ 1.0, CH3OH); IR (KBr): 3415, 2969, 1734, 1648, 1497,
20
(3m)
1457, 1375, 1198, 750, 599 cmꢁ1 1H NMR (300 MHz, DMSO)
; d/
Yield: 87%. Colorless powders. ESI-MS (m/e) 411 [M þ H]þ;
ppm ¼ 9.44 (s, 1 H), 8.75 (d, J ¼ 8.6 Hz, 1 H), 8.58 (s, 1 H), 8.24 (q,
D
[
a]
¼ ꢁ3.1 (c ¼ 1.0, CH3OH); IR (KBr): 3312, 2972, 2883, 1600,
J ¼ 8.4 Hz, 2 H), 7.86 (m, 2 H), 7.32 (m, 5 H), 6.0 (s, 2 H), 5.34 (s, 2 H),
20
1500, 1450, 1379, 1087, 1050, 881, 620 cmꢁ1
;
1H NMR (300 MHz,
4.65 (q, J ¼ 8.4 Hz, 1 H), 2.78 (m, 2 H); 13C NMR (75 MHz, DMSO)
d/
DMSO)
d
/ppm ¼ 9.39 (s, 1 H), 9.08 (d, J ¼ 8.6 Hz, 1 H), 8.54(s, 1 H),
ppm ¼ 172.6,171.8,165.0,152.1,150.8,143.6,138.6,132.1,129.6,128.5,
127.7, 127.6, 127.1, 124.8, 68.7, 52.0, 38.1; Anal. Calcd for C21H19N3O4:
C, 66.83; H, 5.07; N, 11.13. Found C, 66.62; H, 5.23; N, 10.90.
8.24 (q, J ¼ 8.4 Hz, 2 H), 7.86 (m, 2 H), 7.26 (m, 10 H), 5.18 (s, 2 H),
4.94 (q, J ¼ 8.4 Hz, 1 H), 3.32 (m, 2 H); 13C NMR (75 MHz, DMSO)
d/
ppm ¼ 171.6, 164.6, 152.2, 143.4, 137.6, 132.1, 129.1, 128.5, 127.3,
127.1, 124.8, 66.7, 57.0, 36.8; Anal. Calcd for C26H22N2O3: C, 76.08; H,
5.40; N, 6.82. Found C, 75.85; H, 5.56; N, 6.59.
4.4. Bioassay
4.4.1. In vitro anti-proliferation assay
4.3.14. Benzyl (S)-2-(1-hydroxylethyl-1-yl)-2-(isoquinoline-3-
According to the slightly modified procedure of Al-Allaf and
Rashan [13], the in vitro anti-proliferation assays were carried out
by use of 96 microtiter plate cultures and MTT staining. The HL-60
and Hela cells (obtained from Department of Biochemistry, Capital
University of Medical Sciences, final concentration in the growth
medium was 1 ꢀ104/ml) were grown in RPMI-1640 medium
carboxamido)acetate (3n)
Yield: 77%. Colorless powders. ESI-MS (m/e) 455 [M þ H]þ;
D
[
a
]
¼ ꢁ3.2 (c ¼ 1.0, CH3OH); IR (KBr): 3410, 2969, 1734, 1648,
20
1497, 1457, 1440, 1380, 1198, 1112 cmꢁ1; 1H NMR (300 MHz, DMSO)
/ppm ¼ 9.44 (s, 1 H), 8.75 (d, J ¼ 8.6 Hz, 1 H), 8.58 (s, 1 H), 8.24 (q,
d

1680
M. Zheng et al. / European Journal of Medicinal Chemistry 46 (2011) 1672e1681
(containing 10%, v/v) FCS, penicillin (100
m
g/ml) and streptomycin
cell culture inserts, 12 mm in diameter, 3.0 mM in mean pore size).
(100 g/ml) without (for control) or with 3aer (in a series of final
m
Caco-2 cells were grown on filter supports and the integrity of
concentrations ranging from 1 mM to 1 nM). The cultures were
propagated at 37 ꢃC in a humidified atmosphere containing 5% CO2
for 24 h, after the first renew of the growth medium without (for
control) or with 3aer (for test sample) were propagated for another
48 h, after the second renew of the growth medium without (for
control) or with 3aer (for test sample) were propagated for another
4 h. The growth medium was removed and the residue was dried in
monolayers was routinely checked by measurements of trans-
epithelial electrical resistance (approximately 700
U
cm2).
Compounds 2 and 3n, q for evaluation were dissolved in HBSS to
prepare drug solutions at a final concentration of 4 mM. In apical to
basolateral 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 of HBSS (basolateral
compartment). In basolateral to apical direction, transport was
initiated by adding 1.5 ml of the solution of 2 and 3n, q to baso-
lateral compartment and adding 0.5 ml of HBSS as receiving solu-
tion to apical side of the monolayers. The monolayers were
incubated in air at 37 ꢃC and 95% relative humidity. At 30, 60, 90,
and 120 min, samples were withdrawn from the receiving side, and
the concentrations of the samples were determined by HPLC
analysis. The resistance of monolayers was checked at the end of
each test. Apparent permeability coefficients (Papp) were calcu-
lated according to Papp ¼ dQ/dt$1/(A$C0), wherein dQ/dt is the
permeability rate, C0 is the initial concentration in the donor
chamber, and A is the surface area of monolayer (1 cm2).
the air. The dried residues were dissolved in 100 ml of DMSO and the
absorption values of light of the formed purple solutions were
recorded on Bio-rad 450 microplate reader (Biorad, USA). The
inhibited rates were calculated according to I% ¼%(C ꢁ T)/C.
Wherein C presents the absorption value of light of the control and
T presents the absorption value of light of the test sample. The IC50
values were determined via program GWBASIC.EXE.
4.4.2. In vivo anti-tumor assay
Male ICR mice, purchased from Peking University Health Science
Center, were maintained at 21 ꢃC with a natural day/night cycle in
a conventional animal colony. The mice were 10e12 weeks old at
the beginning of the experiments. The tumor used was S180 which
forms solid tumors, when injected subcutaneously. S180 cells for
initiation of subcutaneous tumors were obtained from the ascitic
form of the tumors in mice, which were serially transplanted once
per week. Subcutaneous tumors were implanted by injecting 0.2 ml
of NS containing 1 ꢀ107 viable tumor cells under the skin on the
right oxter. Twenty-four hours after implantation, the tumor-
bearing mice were randomized into 20 experimental groups (10 per
group). All the mice were given a daily i.p. injection of cytarabine
Acknowledgements
This work was completed in the Beijing area major laboratory of
peptide and small molecular drugs, supported by PHR (IHLB, KZ
200810025010), the National Natural Scientific Foundation of China
(20772082, 30801426, 30901843), and Special Project
(2008ZX09401-002) of China.
(positive control, 100
control, 0.2 ml), or 3aer (100
m
mol/kg/day in 0.2 ml of NS), or NS (negative
Appendix A. Supplementary information
mmol/kg/day in 0.2 ml of NS) for 7
consecutive days. Twenty-four hours after the last administration,
all mice were sacrificed by diethyl ether anesthesia and the tumors
were dissected and weighed. The inhibitory rate of tumor growth
was calculated using the equation: Inhibition ¼ (Tumor weight
from negative control mice) ꢁ (Tumor weight from 3aer or cytar-
abine treated mice) O (Tumor weight from negative control mice).
Supplementary information associated with this article can be
found in the online version, at doi:10.1016/j.ejmech.2011.02.017.
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