A class of novel N-(1-methyl-β-carboline-3-carbonyl)-N′- (aminoacid-acyl)-hydrazines: Aromatization leaded design, synthesis, in vitro anti-platelet aggregation/in vivo anti-thrombotic evaluation and 3D QSAR analysis

Article abstract of DOI:10.1016/j.ejmech.2011.09.027

High anti-thrombotic activity of aminoacid modified tetrahydro-β- carbolines was generally correlated with a small proximity of the side chain of the aminoacid residue to the carboline-cycle. This paper explored that the aromatization of the tetrahydro-β-

Full text of DOI:10.1016/j.ejmech.2011.09.027

European Journal of Medicinal Chemistry 46 (2011) 5598e5608
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A class of novel N-(1-methyl-b
-carboline-3-carbonyl)-N0-(aminoacid-acyl)-
hydrazines: Aromatization leaded design, synthesis, in vitro anti-platelet
aggregation/in vivo anti-thrombotic evaluation and 3D QSAR analysis
**
*
Chunyu Li, Xiaoyi Zhang, Ming Zhao , Yuji Wang, Jianhui Wu, Jiawang Liu, Meiqing Zheng, Shiqi Peng
College of Pharmaceutical Sciences, Capital Medical University, No. 10 You An Men Xitoutiao, Beijing 100069, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
High anti-thrombotic activity of aminoacid modified tetrahydro-b-carbolines was generally correlated
Received 30 June 2011
Received in revised form
11 September 2011
Accepted 17 September 2011
Available online 24 September 2011
with a small proximity of the side chain of the aminoacid residue to the carboline-cycle. This paper
explored that the aromatization of the tetrahydro- -carboline-cycle of N-(1-methyl- -tetrahydrocarbo-
line-3-carbonyl)-N0-(aminoacid-acyl)-hydrazines leaded to N-(1-methyl- -carboline-3-carbonyl)-N0-
b
b
b
(aminoacid-acyl)-hydrazines and decreased the proximity of the side chain of the aminoacid residue to the
carboline-cycle. The in vitro activities of inhibiting pig platelet aggregation induced by PAF, ADP, and AA, as
well as the in vivo anti-thrombotic activities of inhibiting rat thrombosis of these aromatized derivatives
Keywords:
were generally higher than that of N-(1-methyl-
hydrazines. The understanding was also obtained from the 3D QSAR analysis.
Ó 2011 Elsevier Masson SAS. All rights reserved.
b
-tetrahydrocarboline-3-carbonyl)-N0-(aminoacid-acyl)-
b-Carboline
Aromatization
Anti-platelet aggregation
Anti-thrombotic activity
3D QSAR
1. Introduction
Pharmacologically, b-carbolines and tetrahydro-b-carbolines
are important indole alkaloids with a wide spectrum of pharma-
Deep vein thrombosis, myocardial infarction, pulmonary
embolism, and stroke have been the most frequent cardiovascular
events, while the intravascular thrombosis has been one of the
most prominent causes of morbidity and mortality [1]. The injury of
blood vessel closely correlates with the arterial thrombosis [2]. If
the vascular endothelium of the injured blood vessel is damaged,
the platelets will adhere to the exposed extracellular matrix [3].
Platelet adhesion is not only the primary event that usually asso-
ciates with the uncontrolled platelet activation and but also
culminates in the intravascular thrombosis [4]. The suppression of
the platelet adhesion and activation, which is achieved in particu-
larly through targeting such secondary regulatory mechanism, is
capable of preventing the thrombosis [5]. Anti-platelet therapy has
a definite role for managing the cardiovascular event [6]. Due to the
most frequently used anti-platelet drugs lack clinical efficacy, there
has been a continuous effort to discover new leads capable of
inhibiting platelet activation and aggregation.
cological actions [7e36]. Harmalol, harmaline, norharmane, har-
mol, harmine and harmane belong to
capable of inhibiting the platelet aggregation induced by collagen,
and had more than 130 M of IC50 value [7]. In the previous paper
N-(1-methyl-
-tetrahydrocarboline-3-carbonyl)-N0-(aminoacid-
b-carboline, were only
m
b
acyl)-hydrazines extended the inhibition to another three inducers,
platelet-activating factor (PAF), adenosine diphosphate (ADP) and
arachidonic acid (AA), and had 0.6e722 mM of IC50 values [37].
In the conformation analysis we used the QSAR module of
Cerius2, explored the in vivo anti-thrombotic activities of aminoacid
modified 3S-tetrahydro-
hexahydropyrazino[10,20:1,6]pyrido [3,4-b]indole- 1,4-dione and
N-(1-methyl-
-tetrahydrocarboline-3-carbonyl)-N0-(aminoacid-acyl)-
b-carbo- line-3-carboxylic acid, (3S,12aS)-
b
hydrazines depended on the proximity of the side chain of the
aminoacid residue to the carboline-cycle, and suggested that
reducing the proximity of the side chain of the aminoacid residue to
the carboline-cycle was beneficial to increasing the in vivo anti-
thrombotic activity [37e39]. Following up this clue, this paper
compared the proximity of the side chain of the aminoacid residue to
* Corresponding author. Tel./fax: þ86 10 8391 1528.
** Corresponding author. Tel./fax: þ86 10 8280 2482.
E-mail addresses: mingzhao@mail.bjmu.edu.cn (M. Zhao), sqpeng@mail.bjmu.
edu.cn (S. Peng).
the
3-carbonyl)-N0-(aminoacid-acyl)-hydrazines with that of their
aromatized derivatives, N-(1-methyl-
-carboline-3-carbonyl)-N0-
carboline-cycle
of
N-(1-methyl-b-tetrahydrocarboline-
b
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.027

C. Li et al. / European Journal of Medicinal Chemistry 46 (2011) 5598e5608
5599
O
R
O
R
H
H
N
N
N
NH
2
N
NH
2
Aromatization
H
H
O
O
NH
N
N
N
H
H
Decrease of the
proximity of
the side chain
to indole-cycle
Fig. 1. QSAR module of Cerius2 derived stereoview of N-(1-methyl-
b
-tetrahydrocarboline-3-carbonyl)-N0-(aminoacid-acyl)-hydrazines and N-(1-methyl-
b-carboline-3 -carbonyl)-
N0- (aminoacid-acyl)-hydrazines.
(aminoacid-acyl)-hydrazines. It was found that the aromatization
leaded to the decrease of the proximity (Fig. 1).
methyl-b
-carboline-3-carbonyl)-N0-(aminoacid-acyl)-hydrazines
(6aep) in 61e92% yields. To ensure the purity, 5aep were purified
on a chromatographic column, while 6aep were purified repeatedly
using ether promoted solidification, and their HPLC purities were
more than 97%. These data demonstrate that the used procedures
In this context, this study prepared sixteen novel N-(1-methyl-
b
-carboline-3-carbonyl)-N0-(aminoacid-acyl)-hydrazines,
evalu-
ated their in vitro activities of inhibiting pig platelet aggregation
induced by PAF, ADP, and AA, tested their in vivo anti-thrombotic
activities of inhibiting rat thrombosis and analyzed their 3D QSAR.
and conditions were suitable for preparing N-(1-methyl-b-caboline-
3-carbonyl)-N0-(aminoacid-acyl)-hydrazines with high quality and
acceptable yields.
2. Results and discussion
2.2. In vitro anti-platelet aggregation of 6aep
2.1. Synthesis of 6aep
The in vitro activities of 6aep (at a series of concentrations
The preparation of N-(1-methyl-
(aminoacid-acyl)-hydrazines (6aep) was carried out according to
the five-step-route depicted in Scheme 1. -Trp was successively
converted into (1S,3S)-1-methyl-1,2,3,4-tetrahydro- - carboline-3-
carboxylic acid (1, 79% yield) via Pictet-Spengler condensation and
(1S,3S)-1-methyl-1,2,3,4-tetrahydro- -carboline-3-carboxylic acid
methyl ester (2, 89% yield) via esterification. The oxidation of 2
resulted in 1-methyl-1,2,3,4-tetrahydro- -carboline-3-carboxylic
acid methyl ester (3) in 65% yield. In the presence of hygrazine
hydrate the hydrazinolysis of 3 provided N-(1-methyl- - carboline-
3-carbonyl)-hydrazine (4) in 62% yield. In the presence of DCC,
b
-carboline-3-carbonyl)-N0-
ranging from 1
aggregation assays. The aggregators were platelet-activating factor
(PAF, final concentration 0.1 M), adenosine diphosphate (ADP, final
concentration 10 M) and arachidonic acid (AA, final concentration
350 M). The in vitro activities of 6aep inhibiting the platelet
mM to 1.5 mM) were identified by the anti-platelet
L
m
b
m
m
b
aggregation induced by PAF, ADP, and AA were represented with
IC50 values and are listed in Table 1. The IC50 values of 4 inhibiting
b
the platelet aggregation induced by AA, PAF and ADP are 32.9
92.8 M and 110.1 M, respectively. The IC50 values of 6aep
inhibiting the platelet aggregation induced by AA, PAF and ADP
range from 0.2 to 9.0 M, 1.1 to 52.9 M, and 20.8 to 74.6 M,
mM,
m
m
b
m
m
m
HOBt and NMM, 4 coupled with Boc-AA to give N-(1-methyl-
b
-
respectively. The in vitro anti-platelet aggregation activities of 6aep
are 1.5e164.5 folds higher than that of 4. It was reported that the
carboline-3-carbonyl)-N0- (Boc-aminoacid-acyl)-hydrazines (5aep,
13%e52% yield). On removing of the Boc group of 5aep gave N-(1-
IC50 values of N-(1-methyl-
b
-tetrahydrocarboline-3-carbonyl)-N0-
CO2H
CO2H
ii
CO2CH3
CO2CH3
i
iii
iii
NH2
NH
NH
N
N
H
N
H
1
N
H
2
N
H
3
CH3
CH3
CH3
iv
CO-NHNH2
CO-NHNH-AA-Boc
CO-NHNH-AA
vi
v
N
N
N
N
N
H
5a-p
N
H
6a-p
H
CH3
CH3
CH3
4
Scheme 1. Synthetic route of N-(1-methyl-b
-caboline-3-carbonyl)-N0-(aminoacid-acyl)-hydrazines. i) CH3CHO and H2SO4; ii) CH3OH and SOCl2; iii) KMnO4; iv) NH2NH2$H2O;
v) DCC, HOBt and NMM; vi) 4N HCl/EtOAc; In 5a and 6a, AA ¼ Gly; In 5b and 6b, AA ¼ Ala; In 5c and 6c, AA ¼ Ile; In 5d and 6d, AA ¼ Leu; In 5e and 6e, AA ¼ Val; In 5f and 6f,
AA ¼ Met; In 5g and 6g, AA ¼ Pro; In 5h and 6h, AA ¼ Tyr; In 5i and 6i, AA ¼ Thr; In 5j and 6j, AA ¼ Ser; In 5k and 6k, AA ¼ Trp; In 5l, AA ¼ His(Boc); In 6l, AA ¼ His; In 5m,
AA ¼ Lys(Boc); In 6m, AA ¼ Lys; In 5n and 6n, AA ¼ Asp(OBzl); In 5o and 6o, AA ¼ Glu(OBzl); In 5p and 6p, AA ¼ Asn.

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C. Li et al. / European Journal of Medicinal Chemistry 46 (2011) 5598e5608
Table 1
Table 3
IC50 of 6aep in vitro platelet aggregation inhibition in the presence of three
Effect of different doses of 6e on the thrombus weight of the rats after oral
aggregators.a
administration.a
Compd.
IC50
(
mM)
Dose
1 nmol/kg
0.1 nmol/kg
0.01 nmol/kg
6e
NS
18.30 ꢀ 1.28b
23.80 ꢀ 1.56c
27.12 ꢀ 1.63d
ADP
PAF
AA
31.87 ꢀ 1.71
4
110.1 ꢀ 9.1
53.3 ꢀ 4.7
58.9 ꢀ 5.3
74.0 ꢀ 6.2
23.0 ꢀ 2.1
20.8 ꢀ 2.1
58.9 ꢀ 4.8
48.7 ꢀ 3.6
74.6 ꢀ 6.1
24.8 ꢀ 3.1
50.9 ꢀ 4.2
28.9 ꢀ 1.9
25.7 ꢀ 1.3
58.9 ꢀ 4.5
81.7 ꢀ 7.8
58.9 ꢀ 4.6
65.8 ꢀ 4.1
92.8 ꢀ 7.9
52.9 ꢀ 4.5
47.4 ꢀ 3.6
5.6 ꢀ 0.5
32.9 ꢀ 2.9
5.2 ꢀ 0.3
6.7 ꢀ 0.2
3.6 ꢀ 0.3
8.2 ꢀ 0.8
0.3 ꢀ 0.01
4.7 ꢀ 0.2
5.8 ꢀ 0.4
1.7 ꢀ 0.1
5.2 ꢀ 0.4
9.0 ꢀ 1.1
8.7 ꢀ 0.9
0.2 ꢀ 0.01
4.1 ꢀ 0.3
3.7 ꢀ 0.2
2.3 ꢀ 0.2
6.2 ꢀ 0.9
a
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
Weight of wet thrombus is represented by X ꢀ SD mg, NS ¼ vehicle, n ¼ 12.
Compared to NS and 0.1 nmol/kg groups p < 0.01.
Compared to NS and 0.01 nmol/kg groups p < 0.01.
Compared to NS group p < 0.01.
b
c
d
1.1 ꢀ 0.2
2.0 ꢀ 0.2
13.4 ꢀ 1.2
44.5 ꢀ 3.5
37.4 ꢀ 2.7
20.9 ꢀ 2.1
52.9 ꢀ 3.9
19.3 ꢀ 1.5
42.5 ꢀ 2.9
52.9 ꢀ 4.3
52.9 ꢀ 3.5
64.3 ꢀ 4.2
39.7 ꢀ 2.8
2.3. In vivo anti-thrombotic activities of 6aep
On extra-corporeal circulation of arterio-veinos cannula model,
the in vivo activities of 6aep were assayed, and the thrombus
weights of the treated rats are listed in Table 2. The data indicated
that the thrombus weights (18.1 mge24.8 mg) of 1 nmol/kg of
6aep treated rats are significantly lower than that (31.9 mg) of
normal saline (NS) treated rats. This means that 1 nmol/kg of
6aep effectively inhibits the rats to form thrombus. Besides, the
thrombus weights (18.1 mge24.8 mg) of 1 nmol/kg of 6aep treated
rats are significantly lower than that (26.7 mg) of 10 nmol/kg of 4
treated rats. This means that the anti-thrombotic activities of 6a-p
are more than 10 folds higher than that of 4. It was reported that
a
n ¼ 6, IC50 is represented with mean ꢀ SD
mM.
(aminoacid-acyl)-hydrazines inhibiting the platelet aggregation
induced by AA, PAF and ADP range from 0.6 to 21.7 M, 1.2 to 506.8
M, and 21.7 to 722.1 M, respectively [37]. This means that the
aromatization leads to a general increase of the in vitro anti-platelet
aggregation activity.
m
m
m
the thrombus weights of 10 nmol/kg of N-(1-methyl-b-tetrahy-
drocarboline-3-carbonyl)-N0-(aminoacid-acyl)-hydrazines treated
rats ranged from 18.6 mg to 24.0 mg (NS, 28.21 mg) [37], which
were substantially equal to that of 1 nmol/kg of 6aep treated rats.
This means that the aromatization leads to a 10-fold increase of the
in vivo anti-thrombotic activity.
To explain the effect of aromatization on the in vitro anti-platelet
aggregation the IC50 values of aromatized compounds (6aep) were
compared with those of non-aromatized compounds [37]. The
statistical analysis indicates that for ADP induced platelet aggre-
gation the IC50 values of 14 aromatized compounds are significantly
lower than that of each respective non-aromatized compound,
while 2 aromatized compounds are significantly higher than that of
each respective non-aromatized compounds. For PAF induced
platelet aggregation the IC50 values of 11 aromatized compounds
are significantly lower than that of each respective non-aromatized
compound, 1 of aromatized compound is equal to that of its non-
aromatized compound, while 4 of aromatized compounds are
significantly higher than that of each respective non-aromatized
compound. For AA induced platelet aggregation the IC50 values of
11 aromatized compounds are significantly lower than that of each
respective non-aromatized compound, while 5 of aromatized
compounds are significantly higher than that of each respective
non-aromatized compound. These statistical data suggested that
a general increase of the in vitro anti-platelet aggregation activity
was achieved with aromatization.
2.4. Dose-dependent in vivo anti-thrombotic activity of 6e
Oral administration of 6e, the most active compound identified
in both in vitro and in vivo assays, was observed at 1 nmol/kg,
0.1 nmol/kg, and 0.01 nmol/kg of doses produce a possible
dose-dependent anti-thrombotic response in rats. The thrombus
weights are listed in Table 3, which demonstrated that the thrombus
weight was progressively increased with dose decrease. Therefore,
6e exhibited dose-dependent anti-thrombotic action.
Table 2
Effect of oral 6aep on the thrombus weight of the rats after oral administration.a
Compd.
Thrombus weight
Compd.
Thrombus weight
NS
4
31.87 ꢀ 1.71
26.66 ꢀ 1.19b
23.70 ꢀ 0.85c
24.39 ꢀ 1.65c
23.12 ꢀ 1.23c
24.49 ꢀ 1.29c
18.30 ꢀ 1.28c
23.43 ꢀ 1.32c
24.37 ꢀ 1.25c
18.56 ꢀ 1.46c
Aspirin
6i
6j
6k
6l
6m
6n
6o
18.86 ꢀ 1.67c
23.65 ꢀ 0.83c
24.78 ꢀ 1.45c
24.56 ꢀ 1.33c
18.10 ꢀ 1.05c
23.30 ꢀ 1.39c
23.23 ꢀ 1.30c
18.80 ꢀ 1.51c
23.97 ꢀ 1.13c
6a
6b
6c
6d
6e
6f
6g
6h
6p
a
Weight of wet thrombus is represented by X ꢀ SD mg, NS ¼ vehicle, n ¼ 12;
Dose of Aspirin: 160
m
mol/kg; Dose of 4: 10 nmol/kg; Dose of 6a-p: 1 nmol/kg.
b
Compared to NS p < 0.001.
Compared to NS and 4 p < 0.001.
c
Fig. 2. Alignment stereoview of 6aep used for molecular field generation.

C. Li et al. / European Journal of Medicinal Chemistry 46 (2011) 5598e5608
5601
atoms and 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 ꢀ30.0 kcal/
mol was set for both electrostatic and steric fields. The total grid
points generated were 982. Though the spatial and structural
descriptors such as dipole moment, polarizability, radius of gyration,
number of rotatable bonds, molecular volume, principal moment of
intertia, AlogP98, number of hydrogen bond donors and acceptors,
and molar refractivity were also considered, only the highest vari-
ance 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 number of components was set to 5. Cross-validationwas
performed with the leave-one-out procedure. PLS analysis was
scaled, with all variables normalized to a variance of 1.0. The MFA
model for the anti-thrombotic activities of 6aec, eei, kep in terms
of the most relevant descriptors including proton, methyl and
hydroxyl anion is expressed by equation (1).
Fig. 3. Graph of tested versus predicted anti-thrombotic activities of 6aep.
2.5. 3D QSAR analysis of 6aep
Thrombus weight ¼ 23:55 ꢁ 0:17ðCH3=678Þ ꢁ 0:18ðCH3=859Þ
À
Á
À
Á
þ 0:079 HOꢁ=597 ꢁ 0:063 HOꢁ=643
2.5.1. Alignment of 6aep
À
Á
À
Á
Establishing the valid 3D-QSAR models a proper alignment
procedure of 6aep was practiced using the target model align
strategy in the align module with Cerius2. Based on the assumption
that each structure of 6aep exhibits activity at the same binding
site of the receptor, they were aligned in a pharmacological active
ꢁ 0:017 HOꢁ=670 þ 0:079 HOꢁ=693
(1)
The correlation of the activities tested on the in vivo thrombo-
genesis model and the activities calculated using equation (1) is
explained by Fig. 3. In equation (1) the correlation coefficient (r)
and square correlation coefficient (r2) are 0.980 and 0.990,
respectively. The tested activities on rat thrombogenesis model and
the calculated activities based on equation (1) are shown in Fig. 3.
The parameters indicated that equation (1) is able to predict the
in vivo activity for the analogs of 6aec, eei, kep.
orientation. To obtain a consistent alignment 1-methyl-b-carbo-
line-3-carbonylhydrazine was selected as the template for super-
posing 6aep. The method used for performing the alignment was
the maximum common subgraph (MCS) [40]. MCS looks at mole-
cules 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 1-methyl-b-carboline-3-carbonylhydrazine that shared by
6aep. This subset was used for the alignment. A rigid fit of atom
pairings was performed to superimpose each structure onto
Equation (1) contains 2 terms from methyl descriptor and 4
terms from hydroxyl anion descriptor. The term of 0.17 (CH3/678)
and 0.18 (CH3/859) have negative coefficient, which means that at
this position hydrophilic group will decrease the thrombus weight.
The term of 0.079 (HOꢁ/597) and 0.079 (HOꢁ/693) have positive
coefficient, which means that at these position electron donating
group will increase the thrombus weight, while term of 0.017 (HOꢁ/
670) has negative coefficient, which means that at this position
electron donating group will decrease the thrombus weight.
As examples Fig. 4 gives two representatives 6k,l, of which 6k
has hydrophobic group near CH3/678 and CH3/859, electron
donating group near HOꢁ/597 and HOꢁ/693, thus has lower in vivo
1-methyl-
6aep is shown in Fig. 2. The alignment stereoview explores that to
superimpose onto 1-methyl- -carboline-3-carbonylhydrazine the
b-carboline-3-carbonylhydrazine. Stereoview of aligned
b
amino acid chain of each structure has to take individual confor-
mation, which will affect on the anti-thrombotic activity.
2.5.2. MFA based Cerius2 QSAR module of 6aep
Molecular field analysis (MFA) was performed for 6aec, eei, kep
using the QSAR module of Cerius2 [41]. A five-step-procedure con-
sisted of generating conformers, energy minimization, matching
Fig. 4. Electrostatic and environments of 6k and 6l within the grid with 3D points of equation (1).

5602
C. Li et al. / European Journal of Medicinal Chemistry 46 (2011) 5598e5608
range of 200e400 nm. 1H NMR and 13C NMR spectra were
recorded on Bruker Advance 300 and 500 spectrometers. FAB-
MS was determined by VG-ZAB-MS high resolution GC/MS/DS
and HP ES-5989x. Optical rotations were determined with
a Schmidt þ 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 cutoff.
Table 4
Predicted and tested thrombus weight of 6d,j treated rats.
Compd.
Thrombus weight (mg)
Predicted
Tested
Error
Error%
6d
6j
23.76
23.88
24.49
24.78
ꢁ0.73
ꢁ0.90
2.98
3.63
4.2. Synthesis
anti-thrombotic activity. While 6l has hydrophilic group near CH3/
678 and electron donating group near HOꢁ/670, thus has higher
in vivo anti-thrombotic activity.
4.2.1. (1S,3S)-1-Methyl-1,2,3,4-tetrahydro-
acid (1)
b-carboline-3-carboxylic
To the mixture of 2.0 g (9.9 mmol) of
L-tryptophane, 200 ml of
2.5.3. Predicting the in vitro anti-thrombotic activity of 6d,j with
equation (1)
water, 0.2 ml of concentrated H2SO4 and 2 ml of aldehyde (40%)
was successively added. The reaction mixture was stirred at room
temperature for 8 h and TLC (CH2Cl2/CH3OH, 10:1) indicates the
The predict power of equation (1) was demonstrated by
comparing the calculated and tested in vitro anti-thrombotic
activity of 6d,j (Table 4). The correlations of predict and test
values are also shown in Fig. 3. The results indicate that equation (1)
rationally gives the thrombus weights for 6d,j with about 0.7 mg
and 0.9 mg of error. The calculated thrombus weights are so
approximate to experimental thrombus weights means that equa-
tion (1) is practical to accurately predict the thrombus weights of N-
complete disappearance of L-tryptophane. The reaction mixture
was adjusted to pH 6e7 with concentrated aqueous solution of
ammonia and kept at 4 ꢃC for 2 h. The formed precipitates were
collected by filtration and washed with water to give 1.8 g (79%)
of the title compound as colorless powder. Mp 287e289 ꢃC; ESI/
MS 231 [M þ H]þ; IR (KBr): 3101e2405, 2962, 2905, 1703, 1624,
1595, 1506, 1453, 1376, 1072, 904 cmꢁ1 1H NMR (500 MHz,
;
(1-methyl-
treated rats.
b
-carboline-3-carbonyl)-N0-(aminoacid-acyl)hydrazines
DMSO-d6)
d
/ppm ¼ 11.92 (s, 1 H), 10.97 (s, 1 H), 9.17 (s, 1 H), 7.45
(d, J ¼ 7.5 Hz, 1 H), 7.36 (t, J ¼ 8.0 Hz, 1 H), 7.10 (t, J ¼ 8.0 Hz, 1 H),
7.01 (t, J ¼ 7.5 Hz, 1 H), 4.22 (q, J ¼ 4.8 Hz, 1 H), 3.66 (dd,
J ¼ 10.5 Hz, J ¼ 5.0 Hz, 1 H), 3.14 (dd, J ¼ 10.5 Hz, J ¼ 2.4 Hz, 1 H),
2.85 (ddd, J ¼ 10.5 Hz, J ¼ 5.0 Hz, J ¼ 2.4 Hz, 1 H), 1.38 (d,
J ¼ 5.0 Hz, 3 H).
3. Conclusions
In conclusion, the aromatization of N-(1-methyl-b-tetrahy-
drocarboline-3-carbonyl)-N0-(aminoacid-acyl)-hydrazines (IC50 of
in vitro anti-platelet aggregation: 0.6e722.1
mM; at 10 nmol/kg
4.2.2. (1S,3S)-1-Methyl-1,2,3,4-tetrahydro-b-carboline-3-carboxylic
dose, in vivo thrombus weights based on a same blank control:
20.96e26.83 mg) successively provided sixteen novel anti-
acid methyl ester (2)
At 0 ꢃC and with stirring to 30 ml of methanol 1.2 ml of SOCl2
was added. This mixture was stirred for 40 min and then was mixed
thrombotic agents, N-(1-methyl-
noacid-acyl)-hydrazines (IC50 of in vitro anti-platelet aggregation:
0.3e81.7 M; at 1 nmol/kg dose, in vivo thrombus weights based on
b
-carboline-3-carbonyl)-N0-(ami-
with 2.0 g (8.7 mmol) of (1S,3S)- 1-methyl-1,2,3,4-Tetrahydro-b-
m
carboline-3-carboxylic acid (1). The reaction mixture was stirred at
room temperature for 12 h and TLC (CH2Cl2/CH3OH, 10:1) indicates
the complete disappearance of 1. The reaction mixture was
neutralized with aqueous sodium carbonate and then kept at 4 ꢃC
for 2 h. The formed precipitates were collected by filtration and
washed with water to give 1.9 g (89%) of the title compound as
yellowing powder. Mp 75e76 ꢃC; ESI-MS (m/z) 245 [M þ H]þ; IR
(KBr): 3400, 3204, 2961, 2903, 2814, 1745, 1622, 1594, 1505, 1453,
a same blank control: 18.10e24.78 mg), profoundly increased the
in vitro anti-platelet aggregation activity and the in vivo anti-
thrombotic activity, and confirmed that the proximity of the side
chain of the amino acid residue to the carboline-cycle of amino acid
modified b-carboline-3-carboxylic acids was a key parameter in the
molecular design.
4. Experimental section
4.1. General
1384, 1062, 895 cmꢁ1; 1H NMR (500 MHz, CDCl3)
d
/ppm ¼ 10.06 (s,
1 H), 7.44 (t, J ¼ 6.4 Hz,1 H), 7.21 (t, J ¼ 8.6 Hz,1 H), 7.13 (d, J ¼ 7.5 Hz,
1 H), 7.02 (d, J ¼ 6.6 Hz, 1 H), 4.31 (q, J ¼ 6.6 Hz, 1 H), 3.79 (s, 3 H),
3.75 (m, J ¼ 6.9 Hz, 1 H), 3.10 (d, J ¼ 11.2 Hz, 1 H), 2.81 (t, J ¼ 11.2 Hz,
1 H), 2.27 (s, 1 H), 1.44 (d, J ¼ 6.0 Hz, 3 H).
The protected amino acids with
L-configuration were
purchased from Sigma Chemical Co. All coupling and depro-
tective reactions were carried out under anhydrous conditions.
Chromatography was performed on Qingdao silica gel H. The
purities of the intermediates and the products were confirmed on
thin layer chromatography TLC (Merck silica gel plates of type 60
F254, 0.25 mm layer thickness) and HPLC. In the HPLC analysis,
a Waters 2695 HPLC system with a Waters Diode Array Detector
was used. The sample was separated on a Symmetry C18 (Waters)
4.2.3. 1-Methyl-
At 0 ꢃC with stirring to the solution of 5.0 g (20.0 mmol) of
(1S, 3S)-1-methyl-1,2,3,4-tetrahydro- -carboline-3-carboxylic acid
b-carboline-3-carboxylic acid methyl ester (3)
b
methyl ester (2) in 50 ml of DMF 4.5 g (28.0 mmol) of potassium
permanganate were added, which took 3 h. The reaction mixture
was stirred at 0 ꢃC for 1 h and then at room temperature for 14 h,
and TLC (CHCl3/CH3OH, 15:1) indicated the complete disappear-
ance of 2. On evaporation the residue was dissolved in 10 ml of
CH3OH. After filtration and evaporation under reduced pressure
3.2 g (65%) of the title compound werþe obtained as yellow powder.
Mp 243e244 ꢃC; ESI/MS 241 [M þ H] ; IR (KBr): 3310, 2954, 2922,
reversed-phase column (5
m
m, 4.6 ꢂ 150 mm). The column
thermostat was maintained at 40 ꢃC. Onto the column 10
m
l of
sample solution was injected for analysis. The mobile phase
consisted of solvent A (CH3OH) and solvent B (H2O). The gradient
elution program consisted of 0e5 min 40% A : 60% B, 5e10 min
45% A : 55% B, 10e15 min 50% A : 50% B and 15e20 min 45% A :
55% B, and the flow rate was 0.6 ml/min. After each run, the
column was washed with methol and equilibrated to initial
conditions for 15 min. The DAD detector was set to a scanning
2901, 2811, 1742, 1600, 1581, 1566, 1450, 1380, 1066, 900 cmꢁ1 1H
;
NMR (500 MHz, CDCl3)
d
/ppm ¼ 9.98 (s, 1 H), 7.43 (d, J ¼ 5.6 Hz,
1 H), 7.33 (d, J ¼ 7.2 Hz, 1 H), 7.16 (d, J ¼ 8.2 Hz, 1 H), 7.14 (t,
J ¼ 7.5 Hz, 1 H), 6.95 (t, J ¼ 6.4 Hz, 1 H), 3.76 (s, 3 H), 2.05 (s, 3 H); 13C
NMR (125 MHz, DMSO-d6)
d/ppm ¼ 167.9, 147.8, 141.6, 135.3, 131.3,

C. Li et al. / European Journal of Medicinal Chemistry 46 (2011) 5598e5608
5603
122.3, 120.0, 115.3, 111.2, 105.3, 51.6, 19.8. Anal. Calcd for
C14H12N2O2: C, 69.99, H, 5.03, N, 11.66. Found: C, 69.78, H, 4.90, N,
11.43.
2.86 (s, 3 H), 1.41 (s, 9 H), 1.32 (d, J ¼ 6.0 Hz, 3 H); 13C NMR (75 MHz,
DMSO-d6)
d
/ppm ¼ 172.1, 163.6, 155.4, 141.7, 141.3, 138.3, 136.5,
128.8, 127.8, 122.6, 121.9, 120.5, 113.1, 112.7, 78.5, 55.4, 28.7, 20.8,
19.0. Anal. Calcd for C21H25N5O4: C, 61.30; H, 6.12; N, 17.02. Found:
C, 61.52; H, 6.37; N, 17.27.
4.2.4. 1-Methyl-
At 60 ꢃC with stirring to the solution of 1.0 g (4.2 mmol) of 1-
methyl- - carboline-3-carboxylic acid methyl ester (3) in 20 ml of
b-carboline-3-carbonylhydrazine (4)
b
4.2.7. N-(1-methyl-b
-caboline-3-carbonyl)-N0-(Boc-isoleucyl)-
CHCl3 and 5 ml of CH3OH 4 ml (80.0 mmol) of hydrazine hydrate was
added dropwise, which took 1 h. The reaction mixture was stirred at
60 ꢃC for another 1 h and TLC (CHCl3/CH3OH,15:1) indicated the
complete disappearance of 3. On evaporation the residue was puri-
fied by silica gel chromatography (CHCl3/CH3OH, 3:1) and 620 mg
(62%) of the title compound was obtained as a colorless powder. Mp
294e295 ꢃC; ESI-MS (m/z) 241 [M þ H]þ; IR (KBr): 3340, 3335, 2924,
2910, 2900, 1620, 1600, 1585, 1562, 1440, 1380, 1066, 900 cmꢁ1; 1H
hydrazine (5c)
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3-carbonyl)-N0-(Boc-glycyl)hydrazine (5a) from 500 mg
(2.08 mmol) of 1-methyl- -carboline-3-carbonylhydrazine (4) and
529 mg (2.29 mmol) of Boc- -Ile 448 mg (48%) of the title
b-
b
L
compound were obtained as yellowing powder. Mp 220e221 ꢃC;
ESI-MS (m/z) 454 [M þ H]þ; ½a D20
¼ ꢁ51.17 (c 1.2, CH3OH); IR
ꢄ
(cmꢁ1): 3272, 2966, 2929, 2872, 1691, 1666, 1621, 1568, 1495, 1462,
1392, 1368, 1348, 1286, 1249, 1168, 1045, 1012, 874, 735; 1H NMR
NMR (500 MHz, DMSO-d6)
d
/ppm ¼ 11.91 (s, 1 H), 8.65 (s, 1 H), 8.33
(d, J ¼ 7.8 Hz,1 H), 7.65 (d, J ¼ 8.1 Hz,1 H), 7.54 (t, J ¼ 7.5 Hz, 1 H), 7.28
(300 MHz, DMSO-d6)
d
/ppm ¼ 12.00 (s, 1 H), 10.23 (s, 2 H), 8.69 (s,
(t, J ¼ 7.5 Hz, 1 H), 4.62 (m, 1 H), 2.80 (s, 3 H); 13C NMR (125 MHz,
1 H), 8.36 (d, J ¼ 7.8 Hz, 1 H), 7.65 (d, J ¼ 7.2 Hz, 1 H), 7.59 (t,
J ¼ 7.5 Hz, 1 H), 7.30 (t, J ¼ 7.5 Hz, 1 H), 6.78 (d, J ¼ 9.0 Hz, 1 H), 4.00
(m, J ¼ 8.7 Hz, 1 H), 2.86 (s, 3 H), 1.75 (m, 1 H), 1.54 (m, 1 H), 1.41 (s,
9 H), 1.15 (s, 1 H), 0.96 (d, J ¼ 6.6 Hz, 3 H), 0.86 (t, J ¼ 7.2 Hz, 3 H); 13C
DMSO-d6)
d
/ppm ¼ 161.6,150.8,140.6,135.2,131.4,122.2,119.6,113.6,
111.1, 106.1, 51.6, 20.0. Anal. Calcd for C13H12N4O: C, 64.99, H, 5.03, N,
23.32. Found: C, 64.77, H, 4.88, N, 23.54.
NMR (75 MHz, DMSO-d6)
d
/ppm ¼ 170.6, 163.7, 155.7, 141.6, 141.2,
4.2.5. N-(1-methyl-
b
-caboline-3-carbonyl)-N0-(Boc-glycyl)-
138.3, 136.5, 128.8, 127.7, 122.6, 121.9, 120.5, 113.1, 112.7, 78.4, 60.2,
45.9, 28.7, 24.8, 20.8, 15.7, 9.3. Anal. Calcd for C24H31N5O4: C, 63.56;
H, 6.89; N, 15.44. Found: C, 63.35; H, 6.74; N, 15.21.
hydrazine (5a)
At 0 ꢃC to the solution of 400 mg (2.29 mmmol) of Boc-Gly,
304 mg (2.25 mmol) of HOBt, 507 mg (2.46 mmol) of DCC and
10 ml of anhydrous THF the solution of 500 mg (2.08 mmol) of 1-
4.2.8. N-(1-methyl-b
-caboline-3-carbonyl)-N0-(Boc-leucyl)-
methyl-b-carboline-3-carbonylhydrazine (4) and 10 ml of anhy-
hydrazine (5d)
drous THF was added. The reaction solution was adjusted to pH 9.5
with 0.6 ml of N-methylmorpholine, stirred at 0 ꢃC for 1 h and at
room temperature for 12 h and TLC (CHCl3/CH3OH,15:1) indicated
the complete disappearance of 4. The reaction mixture was filtered
to remove the resultant precipitates. The filtrate was evaporated
under reduced pressure to remove the solvents. 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 the residue was purified on a chromatographic column
(CH2Cl2/CH3OH, 20:1) to provide 419 mg (50%) of the title compound
as yellowing powder. Mp 219e220 ꢃC; ESI-MS (m/z) 398 [M þ H]þ;
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3-carbonyl)-N0-(Boc-glycyl)hydrazine (5a) from 500 mg
(2.08 mmol) of 1-methyl- -carboline-3-carbonylhydrazine (4) and
529 mg (2.29 mmol) of Boc- -Leu 464 mg (49%) of the title
b-
b
L
compound were obtained as yellowing powder. Mp 186e188 ꢃC;
ESI-MS (m/z) 454 [M þ H]þ; ½a D20
¼ ꢁ15.87 (c 1.2, CH3OH); IR
ꢄ
(cmꢁ1): 3329, 3239, 3043, 2957, 2929, 2871, 1695, 1670, 1625, 1523,
1495, 1446, 1397, 1368, 1348, 1278, 1249, 1168, 1119, 1049, 1025, 878,
730; 1H NMR (300 MHz, DMSO-d6)
d
/ppm ¼ 12.00 (s, 1 H), 10.23 (s,
1 H), 10.21 (s, 1 H), 8.69 (s, 1 H), 8.35 (d, J ¼ 5.1 Hz, 1 H), 7.65 (d,
J ¼ 5.1 Hz, 1 H), 7.59 (t, J ¼ 7.5 Hz, 1 H), 7.30 (t, J ¼ 7.2 Hz, 1 H), 6.93
(d, J ¼ 8.4 Hz,1 H), 4.19 (m, J ¼ 7.8 Hz,1 H), 2.86 (s, 3 H),1.71 (m,1 H),
1.53 (t, J ¼ 6.9 Hz, 2 H), 1.41 (s, 9 H), 0.92 (d, J ¼ 5.1 Hz, 6 H); 13C NMR
½
a 2D0
ꢄ
¼ ꢁ22.72 (c 1.2, CH3OH); IR (cmꢁ1): 3382, 3354, 3108, 3059,
(75 MHz, DMSO-d6)
d
/ppm ¼ 171.9, 163.7, 155.7, 141.6, 141.2, 138.3,
2978, 2929, 1707, 1686, 1621, 1494, 1466, 1446, 1392, 1368, 1348,
136.5, 128.8, 127.7, 122.6, 121.9, 120.5, 113.1, 112.7, 78.4, 51.7, 41.7,
28.7, 24.6, 23.5, 22.1, 20.8. Anal. Calcd for C24H31N5O4: C, 63.56; H,
6.89; N, 15.44. Found: C, 63.75; H, 7.04; N, 15.22.
1282, 1249, 1168, 1049, 735; 1H NMR (300 MHz, DMSO-d6)
d/
ppm ¼ 12.01 (s,1 H),10.21 (s, 1 H),10.17 (s,1 H), 8.69 (s, 1 H), 8.37 (d,
J ¼ 6.0 Hz,1 H), 7.64 (d, J ¼ 3.0 Hz,1 H), 7.60 (t, J ¼ 3.0 Hz,1 H), 7.30 (t,
J ¼ 6.0 Hz,1 H), 7.06 (d, J ¼ 6.0 Hz,1 H), 3.73 (d, J ¼ 6.0 Hz,1 H), 2.86 (s,
4.2.9. N-(1-methyl-b
-caboline-3-carbonyl)-N0-(Boc-valyl)-
hydrazine (5e)
3 H),1.41 (s, 9 H); 13C NMR (75 MHz, DMSO-d6)
156.3, 141.7, 141.2, 138.3, 136.6, 128.8, 127.7, 122.7, 121.9, 120.5, 113.1,
112.7, 78.5, 49.1, 28.7, 20.8. Anal. Calcd for C20H23N5O4: C, 60.44; H,
5.83; N, 17.62. Found: C, 60.21; H, 5.70; N, 17.86.
d
/ppm ¼ 168.7,163.7,
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3- carbonyl)-N0-(Boc-glycyl)hydrazine (5a) from 500 mg
(2.08 mmol) of 1-methyl- - carboline-3-carbonylhydrazine (4) and
497 mg (2.29 mmol) of Boc- -Val 467 mg (51%) of the title
b-
b
L
4.2.6. N-(1-methyl-
b
-caboline-3-carbonyl)-N0-(Boc-alanyl)-
compound were obtained as yellowing powder. Mp 220e221 ꢃC;
hydrazine (5b)
ESI-MS (m/z) 440 [M þ H]þ; ½a D20
¼ ꢁ59.03 (c 1.1, CH3OH); IR
ꢄ
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3- carbonyl)-N0-(Boc-glycyl)hydrazine (5a) from 500 mg
(2.08 mmol) of 1-methyl- - carboline-3-carbonylhydrazine (4) and
433 mg (2.29 mmol) of Boc- -Ala 386 mg (45%) of the title
b
-
(cmꢁ1) 3313, 3260, 2969, 2933, 1686, 1662, 1621, 1531, 1495, 1462,
1392, 1368, 1352, 1298, 1249, 1172, 1045, 1025, 870, 735; 1H NMR
b
(300 MHz, DMSO-d6)
d
/ppm ¼ 12.00 (s, 1 H), 10.23 (s, 1 H), 10.21 (s,
L
1 H), 8.69 (s, 1 H), 8.35 (d, J ¼ 7.8 Hz, 1 H), 7.64 (d, J ¼ 8.1 Hz, 1 H),
7.60 (t, J ¼ 8.1 Hz, 1 H), 7.30 (t, J ¼ 7.8 Hz, 1 H), 6.72 (d, J ¼ 9.3 Hz,
1 H), 3.98 (m, J ¼ 9.0 Hz, 1 H), 2.86 (s, 3 H), 1.99 (m, 1 H), 1.41 (s, 9 H),
compound were obtained as yellowing powder. Mp 157e158 ꢃC;
ESI-MS (m/z) 412 [M þ H]þ; ½a D20
¼ ꢁ74.20 (c 1.1, CH3OH); IR
ꢄ
(cmꢁ1): 3309, 3252, 2978, 2925, 1687, 1621, 1523, 1495, 1450, 1392,
1368, 1348, 1249, 1164, 1049, 861, 726; 1H NMR (300 MHz, DMSO-
1.00 (d, J ¼ 6.9 Hz, 6 H); 13C NMR (75 MHz, DMSO-d6)
d/
ppm ¼ 170.6, 163.8, 155.8, 141.6, 141.2, 138.3, 136.5, 128.8, 127.7,
122.6, 121.9, 120.5, 113.1, 112.7, 78.5, 58.6, 30.9, 28.7, 20.8, 19.7, 18.7.
Anal. Calcd for C23H29N5O4: C, 62.85; H, 6.65; N, 15.93. Found: C,
63.04; H, 6.41; N, 15.70.
d6)
d
/ppm ¼ 12.01 (s, 1 H), 10.22 (s, 2 H), 8.69 (s, 1 H), 8.37 (d,
J ¼ 6.0 Hz, 1 H), 7.67 (d, J ¼ 3.0 Hz, 1 H), 7.60 (t, J ¼ 3.0 Hz, 1 H), 7.30
(t, J ¼ 6.0 Hz, 1 H), 7.01 (d, J ¼ 6.0 Hz, 1 H), 4.19 (m, J ¼ 6.0 Hz, 1 H),

5604
C. Li et al. / European Journal of Medicinal Chemistry 46 (2011) 5598e5608
4.2.10. N-(1-methyl-
hydrazine (5f)
b
-caboline-3-carbonyl)-N0-(Boc-methionyl)-
and 501 mg (2.29 mmol) of Boc-L-Thr 348 mg (38%) of the title
compound were obtained as yellowing powder. Mp 148e149 ꢃC;
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3- carbonyl)-N0-(Boc-glycyl)hydrazine (5a) from 500 mg
(2.08 mmol) of 1-methyl- - carboline-3-carbonylhydrazine (4) and
570 mg (2.29 mmol) of Boc- -Met 336 mg (34%) of the title
b
-
ESI-MS (m/z) 442 [M þ H]þ; ½a D20
¼ ꢁ49.83 (c 1.3, CH3OH); IR
ꢄ
(cmꢁ1): 3293, 2978, 2933, 1695, 1625, 1601, 1568, 1495, 1458, 1397,
1368, 1348, 1303, 1282, 1249, 1168, 1086, 1057, 878, 751, 739; 1H
b
L
NMR (300 MHz, DMSO-d6)
d
/ppm ¼ 12.00 (s, 1 H), 10.22 (s, 1 H),
compound were obtained as yellowing powder. Mp 203e205 ꢃC;
10.20 (s, 1 H), 8.70 (s, 1 H), 8.36 (d, J ¼ 7.8 Hz,1 H), 7.66 (d, J ¼ 8.1 Hz,
1 H), 7.59 (t, J ¼ 7.2 Hz, 1 H), 7.30 (t, J ¼ 7.8 Hz, 1 H), 6.39 (d,
J ¼ 3.9 Hz, 1 H), 4.79 (t, J ¼ 3.6 Hz, 1 H), 4.07 (m, 1 H), 4.02 (m, 1 H),
2.86 (s, 3 H), 1.42 (s, 9 H), 1.19 (d, J ¼ 3.6 Hz, 3 H); 13C NMR (75 MHz,
ESI-MS (m/z) 472 [M þ H]þ; ½a D20
¼ ꢁ36.67 (c 1.2, CH3OH); IR
ꢄ
(cmꢁ1): 3313, 3260, 2978, 2912, 1686, 1653, 1621, 1568, 1523, 1494,
1461, 1446, 1392, 1368, 1348, 1282, 1249, 1168, 1053, 1021, 865, 726;
1H NMR (300 MHz, DMSO-d6)
d/ppm ¼ 12.00 (s, 1 H), 10.23 (s, 1 H),
DMSO-d6)
d
/ppm ¼ 169.7, 163.6, 155.7, 141.7, 141.2, 138.1, 136.5,
10.19 (s, 1 H), 8.69 (s, 1 H), 8.36 (d, J ¼ 8.1 Hz, 1 H), 7.65 (d, J ¼ 8.1 Hz,
1 H), 7.59 (t, J ¼ 7.5 Hz,1 H), 7.29 (t, J ¼ 7.5 Hz,1 H), 7.05 (d, J ¼ 8.1 Hz,
1 H), 4.19 (m, 1 H), 2.86 (s, 3 H), 2.59 (t, J ¼ 7.5 Hz, 1 H), 2.09 (s, 3 H),
128.8, 127.7, 122.6, 121.9, 120.5, 113.1, 112.7, 78.7, 67.6, 65.3, 28.6,
20.8. Anal. Calcd for C22H27N5O5: C, 59.85; H, 6.16; N, 15.86. Found:
C, 59.64; H, 6.01; N, 15.63.
1.95 (m, 2 H), 1.41 (s, 9 H); 13C NMR (75 MHz, DMSO-d6)
d/
ppm ¼ 171.1, 163.9, 155.7, 141.6, 141.2, 138.3, 136.5, 128.8, 127.7,
122.6, 121.9, 120.5, 113.1, 112.7, 78.6, 52.7, 32.7, 29.9, 28.7, 20.8, 15.6.
Anal. Calcd for C23H29N5O4S: C, 58.58; H, 6.20; N, 14.85. Found: C,
58.37; H, 6.04; N, 14.62.
4.2.14. N-(1-methyl-b
-caboline-3-carbonyl)-N0-(Boc-seryl)-
hydrazine (5j)
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3- carbonyl)-N0-(Boc-glycyl)hydrazine (5a) from 500 mg
(2.08 mmol) of 1-methyl- - carboline-3-carbonylhydrazine (4) and
469 mg (2.29 mmol) of Boc- -Ser 324 mg (37%) of the title
b-
b
4.2.11. N-(1-methyl-
b
-caboline-3-carbonyl)-N0-(Boc-prolyl)-
L
hydrazine (5g)
compound were obtained as yellowing powder. Mp 144e146 ꢃC;
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3- carbonyl)-N0-(Boc-glycyl)hydrazine (5a) from 500 mg
(2.08 mmol) of 1-methyl- - carboline-3-carbonylhydrazine (4) and
492 mg (2.29 mmol) of Boc- -Pro 476 mg (52%) of the title
b
-
ESI-MS (m/z) 428 [M þ H]þ; ½a D20
¼ ꢁ16.10 (c 1.1, CH3OH); IR
ꢄ
(cmꢁ1): 3284, 2982, 2933, 1695, 1625, 1597, 1572, 1495, 1462, 1392,
1368, 1348, 1303, 1282, 1249, 1168, 1061, 898, 755, 735; 1H NMR
b
L
(300 MHz, DMSO-d6)
d
/ppm ¼ 12.02 (s, 1 H), 10.30 (s, 1 H), 10.24 (s,
compound were obtained as yellowing powder. Mp 227e228 ꢃC;
1 H), 8.70 (s, 1 H), 8.36 (d, J ¼ 4.8 Hz, 1 H), 7.66 (d, J ¼ 4.8 Hz, 1 H),
7.59 (t, J ¼ 4.5 Hz, 1 H), 7.30 (t, J ¼ 4.5 Hz, 1 H), 6.75 (d, J ¼ 5.1 Hz,
1 H), 4.91 (t, J ¼ 3.6 Hz,1 H), 4.21 (dd, J ¼ 7.8 Hz, J ¼ 3.6 Hz,1 H), 3.66
ESI-MS (m/z) 438 [M þ H]þ; ½a D20
¼ ꢁ63.25 (c 1.1, CH3OH); IR
ꢄ
(cmꢁ1): 3382, 3301, 3244, 3019, 2974, 2929, 1707, 1683, 1629, 1572,
1523, 1486, 1458, 1442, 1405, 1368, 1352, 1303, 1249, 1168, 1115,
(m, 2 H), 2.86 (s, 3 H), 1.41 (s, 9 H); 13C NMR (75 MHz, DMSO-d6)
d/
1086, 890, 751; 1H NMR (300 MHz, DMSO-d6)
d/ppm ¼ 12.00 (s,
ppm ¼ 169.7, 163.6, 155.6, 141.7, 141.2, 138.1, 136.5, 128.8, 127.7,
122.6, 121.9, 120.5, 113.1, 112.7, 78.6, 62.6, 56.0, 28.7, 20.8. Anal.
Calcd for C21H25N5O5: C, 59.01; H, 5.90; N, 16.38. Found: C, 58.82; H,
5.75; N, 16.61.
1 H), 10.21 (s, 1 H), 10.14 (s, 1 H), 8.72 (s, 1 H), 8.37 (d, J ¼ 7.5 Hz, 1 H),
7.65 (d, J ¼ 8.1 Hz,1 H), 7.59 (t, J ¼ 7.2 Hz,1 H), 7.29 (t, J ¼ 7.2 Hz,1 H),
4.27 (m, 1 H), 3.43 (m, 1 H), 3.30 (m, 1 H), 2.88 (s, 3 H), 2.19 (m, 1 H),
1.80 (m, 3 H), 1.41 (s, 9 H); 13C NMR (75 MHz, DMSO-d6)
d/
ppm ¼ 171.1,163.6, 157.1, 153.7, 141.6,141.2, 138.3,136.5, 128.8, 127.7,
122.6, 121.9, 120.5, 113.1, 112.7, 79.1, 58.7, 47.9, 31.5, 28.6, 24.9, 20.8.
Anal. Calcd for C23H27N5O4: C, 63.14; H, 6.22; N, 14.63. Found: C,
63.36; H, 6.38; N, 14.41.
4.2.15. N-(1-methyl-b
-caboline-3-carbonyl)-N0-(Boc-trptophanyl)-
hydrazine (5k)
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3- carbonyl)-N0-(Boc-glycyl)hydrazine (5a) from 500 mg
(2.08 mmol) of 1-methyl- - carboline-3-carbonylhydrazine (4) and
696 mg (2.29 mmol) of Boc- -Trp 445 mg (41%) of the title
b-
b
4.2.12. N-(1-methyl-
hydrazine (5h)
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3- carbonyl)-N0-(Boc-glycyl)hydrazine (5a) from 500 mg
(2.08 mmol) of 1-methyl- - carboline-3-carbonylhydrazine (4) and
644 mg (2.29 mmol) of Boc-
b
-caboline-3-carbonyl)-N0-(Boc-tyrosyl)-
L
compound were obtained as yellowing powder. Mp 169e171 ꢃC;
b
-
ESI-MS (m/z) 527 [M þ H]þ; ½a D20
¼ ꢁ22.43 (c 1.2, CH3OH); IR
ꢄ
(cmꢁ1): 3427, 3407, 3387, 3313, 3051, 2978, 2929, 1691, 1666, 1625,
1601, 1572, 1531, 1495, 1458, 1392, 1368, 1352, 1274, 1249, 1172,
b
L-Tyr 422 mg (40%) of the title
1094, 1049, 1045, 1008, 739; 1H NMR (300 MHz, DMSO-d6)
d/
ꢃ
compound were obtained as yellowing powder. Mp 164e166 C;
ppm ¼ 12.02 (s, 1 H), 10.84 (s, 1 H), 10.47 (s, 1 H), 10.24 (s, 1 H), 8.73
(s, 1 H), 8.38 (d, J ¼ 3.0 Hz, 1 H), 7.72 (d, J ¼ 6.0 Hz, 1 H), 7.68 (t,
J ¼ 3.0 Hz, 1 H), 7.60 (t, J ¼ 3.0 Hz, 1 H), 7.35 (d, J ¼ 3.0 Hz, 1 H), 7.33
(d, J ¼ 3.0 Hz, 1 H), 7.29 (s, 1 H), 7.09 (t, J ¼ 3.0 Hz, 1 H), 7.03 (t,
J ¼ 3.0 Hz, 1 H), 6.80 (t, J ¼ 3.0 Hz, 1 H), 4.44 (m, 1 H), 3.24 (dd,
J ¼ 9.0 Hz, J ¼ 3.0 Hz, 1 H), 3.03 (t, J ¼ 6.0 Hz, 1 H), 2.88 (s, 3 H), 1.34
ESI-MS (m/z) 504 [M þ H]þ; ½a D20
ꢄ
¼ ꢁ9.77 (c 1.1, CH3OH); IR (cmꢁ1
)
3305, 2978, 2925, 1691, 1670, 1621, 1597, 1519, 1495, 1462, 1446,
1388, 1368, 1348, 1249, 1168, 1098, 1049, 1021, 751, 739; 1H NMR
(300 MHz, DMSO-d6)
d/ppm ¼ 12.05 (s, 1 H), 10.40 (s, 1 H), 10.25 (s,
1 H), 9.18 (s, 1 H), 8.71 (s, 1 H), 8.37 (d, J ¼ 7.8 Hz, 1 H), 7.66 (d,
J ¼ 8.1 Hz,1 H), 7.59 (t, J ¼ 7.2 Hz,1 H), 7.30 (t, J ¼ 7.5 Hz,1 H), 7.13 (d,
J ¼ 8.1 Hz, 2 H), 6.90 (d, J ¼ 8.7 Hz, 1 H), 6.68 (d, J ¼ 8.4 Hz, 2 H), 4.28
(m, 1 H), 4.07 (m, 1 H), 3.02 (m, 1 H), 2.86 (m, 1 H), 1.33 (s, 9 H); 13C
(s, 9 H); 13C NMR (75 MHz, DMSO-d6)
d
/ppm ¼ 171.2, 163.6, 155.6,
141.7,141.2,138.3,136.5,128.8,127.9,127.8,124.8,124.4,122.6, 121.9,
121.2, 120.9, 120.5, 119.0, 118.6, 113.7, 113.1, 112.7, 111.7, 110.7, 110.4,
78.4, 49.1, 28.6, 20.8. Anal. Calcd for C29H30N6O4: C, 66.14; H, 5.74;
N, 15.96. Found: C, 66.33; H, 5.59; N, 15.74.
NMR (75 MHz, DMSO-d6)
d
/ppm ¼ 171.2, 163.6, 156.2, 155.5, 141.7,
141.2, 138.3, 136.5, 130.7, 128.8, 127.7, 122.6, 121.9, 120.5, 115.3, 113.1,
112.7, 78.4, 56.5, 37.5, 28.6, 20.8. Anal. Calcd for C27H29N5O5: C,
64.40; H, 5.80; N, 13.91. Found: C, 64.59; H, 5.95; N, 13.69.
4.2.16. N-(1-methyl-b
-caboline-3-carbonyl)-N0-(di-Boc-histidyl)-
hydrazine (5l)
4.2.13. N-(1-methyl-
hydrazine (5i)
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3- carbonyl)-N0-(Boc-glycyl)hydrazine (5a) from 500 mg
(2.08 mmol) of 1-methyl- - carboline-3-carbonylhydrazine (4)
b
-caboline-3-carbonyl)-N0-(Boc-threonyl)-
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3- carbonyl)-N0-(Boc-glycyl)hydrazine (5a) from 500 mg
(2.08 mmol) of 1-methyl- - carboline-3-carbonylhydrazine (4) and
813 mg (2.29 mmol) of Boc- -His(Boc) 524 mg (44%) of the title
compound were obtained as yellowing powder. Mp 184e185 ꢃC;
b-
b
-
b
L
b

C. Li et al. / European Journal of Medicinal Chemistry 46 (2011) 5598e5608
5605
ESI-MS (m/z) 578 [M þ H]þ; ½a D20
ꢄ
¼ ꢁ39.90 (c 1.1, CH3OH); IR
(m, 6 H), 7.02 (d, J ¼ 8.1 Hz, 1 H), 5.13 (s, 2 H), 4.16 (m, 1 H), 2.86 (s,
(cmꢁ1) 3284, 2978, 2929, 1756, 1690, 1666, 1625, 1596, 1568, 1490,
1457, 1396, 1372, 1348, 1282, 1249, 1164, 1053, 1008, 845, 775, 755,
3 H), 2.58 (m, 2 H), 1.97 (m, 2 H), 1.41 (s, 9 H); 13C NMR (75 MHz,
DMSO-d6)
d
/ppm ¼ 172.8, 170.9, 163.9, 155.6, 141.6, 141.2, 138.3,
739; 1H NMR (300 MHz, DMSO-d6)
d
/ppm ¼ 12.00 (s, 1 H), 10.31 (s,
136.7, 128.9, 128.4, 127.7, 122.6, 121.8, 120.5, 113.2, 112.7, 78.6, 65.9,
52.5, 30.4, 28.7, 28.1, 20.8. Anal. Calcd for C30H33N5O6: C, 64.39; H,
5.94; N, 12.51. Found: C, 64.28; H, 5.80; N, 12.29.
1 H), 10.21 (s, 1 H), 8.69 (s, 1 H), 8.35 (d, J ¼ 7.8 Hz, 1 H), 8.15 (s, 1 H),
7.66 (d, J ¼ 8.1 Hz, 1 H), 7.59 (t, J ¼ 7.5 Hz, 1 H), 7.30 (t, J ¼ 7.5 Hz,
2 H), 6.96 (d, J ¼ 8.7 Hz, 1 H), 4.42 (m, 1 H), 2.95 (m, 1 H), 2.86 (s,
4 H), 1.57 (s, 9 H), 1.37 (s, 9 H); 13C NMR (75 MHz, DMSO-d6)
d/
4.2.20. N-(1-methyl-b
-caboline-3-carbonyl)-N0-(Boc-glutaminyl)-
ppm ¼ 170.8, 163.7, 155.6, 147.2, 141.7, 141.2, 139.9, 138.3, 137.0,
136.5, 128.8, 127.7, 122.6, 121.9, 120.5, 114.9, 113.1, 112.7, 85.6, 78.6,
54.2, 31.5, 28.6, 20.8. Anal. Calcd for C29H35N7O6: C, 60.30; H, 6.11;
N, 16.97. Found: C, 60.09; H, 6.27; N, 16.74.
hydrazine (5p)
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3- carbonyl)-N0-(Boc-glycyl)hydrazine (5a) from 500 mg
(2.08 mmol) of 1-methyl- - carboline-3-carbonylhydrazine (4)
and 563 mg (2.29 mmol) of Boc- -Gln 130 mg (13%) of the title
b-
b
L
4.2.17. N-(1-methyl-
b
-caboline-3-carbonyl)-N0-(di-Boc-lysyl)-
compound were obtained as yellowing powder. Mp 215e216 ꢃC;
hydrazine (5m)
ESI-MS (m/z) 469 [M þ H]þ; ½a D20
¼ ꢁ21.61 (c 1.1, CH3OH); IR
ꢄ
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3- carbonyl)-N0-(Boc-glycyl)hydrazine (5a) from 500 mg
(2.08 mmol) of 1-methyl- - carboline-3-carbonylhydrazine (4) and
792 mg (2.29 mmol) of Boc- -Lys(Boc) 392 mg (33%) of the title
b
-
(cmꢁ1): 3423, 3333, 3268, 2978, 2921, 2851, 1691, 1646, 1519, 1499,
1450, 1392, 1368, 1352, 1319, 1278, 1249, 1172, 1057, 865, 722; 1H
b
NMR (300 MHz, DMSO-d6)
d
/ppm ¼ 12.01 (s, 1 H), 10.15 (s, 2 H),
L
8.69 (s, 1 H), 8.36 (d, J ¼ 7.5 Hz, 1 H), 7.65 (d, J ¼ 8.1 Hz, 1 H), 7.59 (t,
J ¼ 7.5 Hz, 1 H), 7.29 (m, 2 H), 6.97 (d, J ¼ 8.1 Hz, 1 H), 6.57 (s, 1 H),
4.10 (m, 1 H), 2.86 (s, 3 H), 2.25 (m, 2 H), 1.96 (m, 1 H), 1.73 (m, 1 H),
compound were obtained as yellowing powder. Mp 125e127 ꢃC;
ESI-MS (m/z) 569 [M þ H]þ; ½a D20
¼ ꢁ34.07 (c 1.3, CH3OH); IR
ꢄ
(cmꢁ1): 3329, 3268, 2978, 2929, 2864, 1687, 1625, 1601, 1519, 1495,
1458, 1392, 1368, 1348, 1282, 1249, 1168, 1053, 1012, 878, 739; 1H
1.41 (s, 9 H); 13C NMR (75 MHz, DMSO-d6)
d
/ppm ¼ 174.3, 171.1,
163.7, 155.6, 141.7, 141.2, 138.3, 136.5, 128.8, 127.7, 122.6, 121.8,
120.5, 113.1, 112.7, 78.6, 53.0, 31.9, 28.7, 20.8. Anal. Calcd for
C23H28N6O5: C, 58.96; H, 6.02; N, 17.94. Found: C, 58.74; H, 5.86; N,
17.72.
NMR (300 MHz, DMSO-d6)
d
/ppm ¼ 12.00 (s, 1 H), 10.20 (s, 1 H),
8.35 (d, J ¼ 7.8 Hz, 1 H), 7.65 (d, J ¼ 7.8 Hz, 1 H), 7.65 (t, J ¼ 7.2 Hz,
1 H), 7.29 (d, J ¼ 7.2 Hz, 1 H), 6.89 (d, J ¼ 8.7 Hz, 1 H), 6.79 (m, 1 H),
4.06 (m, 1 H), 2.93 (m, 2 H), 2.86 (s, 3 H), 1.66 (m, 2 H), 1.38 (m,
24 H); 13C NMR (75 MHz, DMSO-d6)
d
/ppm ¼ 171.6, 163.6, 156.1,
4.2.21. N-(1-methyl-
At 0 ꢃC to the solution of 100 mg (0.252 mmol) of N-[(1S,3S)-1-
methyl-
- caboline-3-carbonyl]-N0-(Boc-glycyl)-hydrazine (5a) in
b
-caboline-3-carbonyl)-N0-glycylhydrazine (6a)
155.7, 141.6, 141.2, 138.3, 136.5, 128.8, 127.8, 122.6, 121.9, 120.5, 113.1,
112.7, 78.5, 77.8, 60.2, 53.3, 32.5, 29.7, 28.7, 23.1, 20.8. Anal. Calcd for
C29H40N6O6: C, 61.25; H, 7.09; N, 14.78. Found: C, 61.06; H, 6.93; N,
14.99.
b
6 ml hydrogen chloride/ethyl acetate (4N) were added dropwise.
The reaction solution was stirred at 0 ꢃC for 2 h and (CHCl3/CH3OH,
10:1) indicated the complete disappearance of 5a. The reaction
mixture was evaporated under reduced pressure. The residue was
dissolved in 5 ml of ethyl acetate and then evaporated under
reduced pressure. This procedure was repeated for three times to
remove the remained hydrogen chloride. The residue was solidified
in 20 ml of anhydrous ether to provide 687 mg (92%) of the title
compound as yellowing powder. Mp 231e233 ꢃC; ESI-MS (m/z) 298
4.2.18. N-(1-methyl-
aspartyl)]-hydrazine (5n)
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3- carbonyl)-N0-(Boc-glycyl)hydrazine (5a) from 500 mg
(2.08 mmol) of 1-methyl- - carboline-3-carbonylhydrazine (4) and
740 mg (2.29 mmol) of Boc- -Asp(OBzl) 348 mg (31%) of the title
b b-OBzl-
-caboline-3-carbonyl)-N0-[Boc-(
b
-
b
L
compound were obtained as yellowing powder. Mp 130e132 ꢃC;
[M þ H]þ; ½a 2D0
ꢄ
¼ 9.98 (c 1.2, CH3OH); IR (cmꢁ1): 3423, 2998, 2962,
ESI-MS (m/z) 546 [M þ H]þ; ½a D20
ꢄ
¼ ꢁ22.32 (c 1.2, CH3OH); IR
2925, 2855, 1691, 1625, 1511, 1364, 1245, 755; 1H NMR (300 MHz,
(cmꢁ1): 3309, 3260, 3064, 3031, 2982, 2929, 1719, 1691, 1662, 1625,
1601, 1572, 1499, 1462, 1392, 1372, 1352, 1278, 1249, 1164, 1049,
DMSO-d6)
d
/ppm ¼ 12.72 (s, 1 H), 10.85 (s, 1 H), 8.99 (s, 1 H), 8.40
(m, 4 H), 7.72 (d, J ¼ 8.1 Hz, 1 H), 7.67 (t, J ¼ 7.8 Hz, 1 H), 7.37 (t,
1025, 865, 751, 739; 1H NMR (300 MHz, DMSO-d6)
d/ppm ¼ 11.99 (s,
J ¼ 7.2 Hz, 1 H), 3.74 (m, 2 H), 2.99 (s, 3 H); 13C NMR (75 MHz,
1 H), 10.17 (s, 1 H), 8.35 (d, J ¼ 7.8 Hz, 1 H), 7.65 (d, J ¼ 7.8 Hz, 1 H),
7.59 (t, J ¼ 7.2 Hz, 1 H), 7.36 (m, 6 H), 7.19 (d, J ¼ 8.1 Hz, 1 H), 5.14 (s,
2 H), 4.56 (m, 1 H), 2.91 (m, 2 H), 2.86 (s, 3 H), 2.71 (m, 2 H), 1.41 (s,
DMSO-d6)
d
/ppm ¼ 170.2, 165.7, 162.2, 142.5, 142.1, 136.4, 130.3,
128.9, 123.0, 121.5, 121.3, 114.6, 113.3, 65.4, 19.0. Anal. Calcd for
C15H15N5O2: C, 60.60; H, 5.09; N, 23.56. Found: 60.41; H, 4.93; N,
23.33.
9 H); 13C NMR (75 MHz, DMSO-d6)
d
/ppm ¼ 170.4, 163.7, 155.6,
141.6, 141.2, 138.3, 136.5, 128.8, 127.7, 127.0, 122.6, 121.8, 120.5, 113.1,
112.7, 78.8, 66.1, 50.1, 37.1, 28.7, 20.8. Anal. Calcd for C29H31N5O6: C,
63.84; H, 5.73; N, 12.84. Found: C, 63.62; H, 5.58; N, 13.06.
4.2.22. N-(1-methyl-
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3-carbonyl)-N0-glycylhydrazine (6a) from 100 mg
(0.243 mmol) of N-[(1S,3S)-1-methyl-
-caboline-3-carbonyl]-N0-
b
-caboline-3-carbonyl)-N0-alanylhydrazine (6b)
b
-
4.2.19. N-(1-methyl-
glutamoyl)]-hydrazine (5o)
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3-carbonyl)-N0-(Boc-glycyl)hydrazine (5a) from 500 mg
(2.08 mmol) of 1-methyl- -carboline-3-carbonylhydrazine (4) and
771 mg (2.29 mmol) of Boc- -Glu(OBzl) 351 mg (30%) of the title
b
-caboline-3-carbonyl)-N0-[Boc-(
g
-OBzl-
b
(Boc-alanyl)-hydrazine (5b) 71 mg (93%) of the title compound
b
-
were obtained as yellowing powder. Mp 228e229 ꢃC; ESI-MS (m/z)
312 [M þ H]þ; ½a 2D0
ꢄ
¼ ꢁ14.57 (c 1.1, CH3OH); IR (cmꢁ1): 3309, 3252,
b
2978, 2925, 1687, 1621, 1523, 1495, 1450, 1392, 1368, 1348, 1249,
L
1164, 1049, 861, 726; 1H NMR (300 MHz, DMSO-d6)
d
/ppm ¼ 12.53
compound were obtained as yellowing powder. Mp 191e193 ꢃC;
(s, 1 H), 10.81 (s, 1 H), 10.74 (s, 1 H), 8.40 (m, 4 H), 7.72 (d, J ¼ 8.1 Hz,
1 H), 7.67 (t, J ¼ 7.8 Hz, 1 H), 7.37 (t, J ¼ 7.2 Hz, 1 H), 4.02 (m,
J ¼ 7.2 Hz, 1 H), 2.95 (s, 3 H), 1.50 (d, J ¼ 6.9 Hz, 3 H); 13C NMR
ESI-MS (m/z) 560 [M þ H]þ; ½a D20
¼ ꢁ21.10 (c 1.3, CH3OH); IR
ꢄ
(cmꢁ1): 3329, 3276, 3064, 3031, 3006, 2982, 2929, 1723, 1687, 1662,
1625, 1593, 1523, 1491, 1462, 1450, 1392, 1368, 1348, 1286, 1249,
(75 MHz, DMSO-d6)
d
/ppm ¼ 169.2, 168.9, 162.9, 142.0, 136.3, 129.8,
1168, 1082, 1066, 1025, 878, 730; 1H NMR (300 MHz, DMSO-d6)
d/
128.6, 122.9, 121.5, 121.2, 114.1, 113.1, 48.0, 19.7, 17.8. Anal. Calcd
for C16H17N5O2: C, 61.72; H, 5.50; N, 22.49. Found: C, 61.50; H, 5.35;
N, 22.71.
ppm ¼ 11.99 (s,1 H),10.23 (s,1 H), 10.19 (s,1 H), 8.69 (s, 1 H), 8.35 (d,
J ¼ 7.8 Hz, 1 H), 7.65 (d, J ¼ 7.8 Hz, 1 H), 7.59 (t, J ¼ 7.5 Hz, 1 H), 7.36

5606
C. Li et al. / European Journal of Medicinal Chemistry 46 (2011) 5598e5608
4.2.23. N-(1-methyl-
(6c)
b
-caboline-3-carbonyl)-N0-isoleucylhydrazine
122.9, 121.4, 114.5, 113.2, 51.0, 31.6, 28.2, 19.2, 14.9. Anal. Calcd for
C18H21N5O2S: C, 58.20; H, 5.70; N, 18.85. Found: C, 58.01; H, 5.55; N,
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3-carbonyl)-N0-glycylhydrazine (6a) from 100 mg
(0.221 mmol) of N-(1-methyl-
-caboline-3-carbonyl)-N0-(Boc-iso-
b
-
18.62.
b
4.2.27. N-(1-methyl-b
-caboline-3-carbonyl)-N0-prolylhydrazine (6g)
leucyl)-hydrazine (5c) 70 mg (90%) of the title compound were
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3-carbonyl)-N0-glycylhydrazine (6a) from 100 mg
(0.212 mmol) of N-(1-methyl-
-caboline-3-carbonyl)-N0-(Boc-
b-
obtained as yellowing powder. Mp 211e212 ꢃC; ESI-MS (m/z) 354
[M þ H]þ; ½a 2D0
ꢄ
¼ 10.45 (c 1.1, CH3OH); IR (cmꢁ1): 3440, 3072, 2966,
b
2921, 2876, 1715, 1674, 1634, 1507, 1364, 1245, 1217, 1057, 755; 1H
prolyl)-hydrazine (5g) 71 mg (90%) of the title compound were
NMR (300 MHz, DMSO-d6)
d
/ppm ¼ 12.72 (s, 1 H), 10.89 (s, 1 H),
obtained as yellowing powder. Mp 242e243 ꢃC; ESI-MS (m/z) 372
10.79 (s,1 H), 8.40 (m, 4 H), 7.75 (d, J ¼ 8.1 Hz,1 H), 7.67 (t, J ¼ 7.2 Hz,
1 H), 7.37 (t, J ¼ 7.5 Hz, 1 H), 3.82 (m, 1 H), 3.00 (s, 3 H), 1.94 (m, 1 H),
1.68 (m, 1 H), 1.32 (m, 1 H), 1.05 (d, J ¼ 7.5 Hz, 3 H), 0.97 (t, J ¼ 7.5 Hz,
[M þ H]þ; ½a 2D0
ꢄ
¼ 25.17 (c 1.1, CH3OH); IR (cmꢁ1): 3427, 3080, 2978,
2916, 2843, 1711, 1679, 1629, 1511, 1364, 1249, 1221, 869, 755; 1H
NMR (300 MHz, DMSO-d6)
d
/ppm ¼ 12.75 (s,1 H),11.06 (s,1 H), 9.09
3 H); 13C NMR (75 MHz, DMSO-d6)
d
/ppm ¼ 167.2, 166.9, 161.7,
(s,1 H), 8.59 (m, 3 H), 8.37 (d, J ¼ 8.1 Hz,1 H), 7.75 (d, J ¼ 8.1 Hz,1 H),
7.67 (t, J ¼ 8.1 Hz, 1 H), 7.39 (t, J ¼ 7.5 Hz, 1 H), 4.14 (m, 1 H), 3.01 (s,
3 H), 2.71 (m, 2 H), 2.21 (m, 2 H), 2.12 (s, 3 H); 13C NMR (75 MHz,
142.8, 142.2, 136.1, 130.6, 129.2, 123.0, 121.7, 121.2, 114.9, 113.4, 55.7,
36.9, 24.6, 18.6, 14.6, 11.8. Anal. Calcd for C19H23N5O2: C, 64.57; H,
6.56; N, 19.82. Found: C, 64.35; H, 6.41; N, 19.61.
DMSO-d6)
d
/ppm ¼ 167.8, 162.5, 142.4, 142.1, 136.2, 130.2, 128.8,
122.9, 121.4, 114.5, 113.2, 51.0, 31.6, 28.2, 19.2, 14.9. Anal. Calcd for
C18H21N5O2S: C, 58.20; H, 5.70; N, 18.85. Found: C, 58.41; H, 5.86; N,
19.07.
4.2.24. N-(1-methyl-
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3-carbonyl)-N0-glycylhydrazine (6a) from 100 mg
(0.221 mmol) of N-(1-methyl-
-caboline-3-carbonyl)-N0-(Boc-
b
-caboline-3-carbonyl)-N0-leucylhydrazine (6d)
b
-
b
4.2.28. N-(1-methyl-b
-caboline-3-carbonyl)-N0-tyrosylhydrazine
leucyl)-hydrazine (5d) 70 mg (90%) of the title compound were
(6h)
obtained as yellowing powder. Mp 238e239 ꢃC; ESI-MS (m/z) 354
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3-carbonyl)-N0-glycylhydrazine (6a) from 100 mg
(0.199 mmol) of N-(1-methyl-
-caboline-3-carbonyl)-N0-(Boc-
b-
[M þ H]þ; ½a 2D0
ꢄ
¼ 45.97 (c 1.1, CH3OH); IR (cmꢁ1): 3439, 3411,
3068, 2961, 2929, 2867, 1715, 1679, 1629, 1507, 1364, 1249, 1217,
b
1029, 755; 1H NMR (300 MHz, DMSO-d6)
d
/ppm ¼ 12.94 (s, 1 H),
tyrosyl)-hydrazine (5h) 72 mg (89%) of the title compound were
11.08 (s,1 H), 9.12 (s,1 H), 8.40 (m, 4 H), 7.76 (d, J ¼ 8.1 Hz, 1 H), 7.67
(t, J ¼ 8.1 Hz, 1 H), 7.37 (t, J ¼ 7.2 Hz, 1 H), 3.92 (m, 1 H), 2.98 (s, 3 H),
1.94 (m, 1 H), 1.68 (m, 2 H), 0.96 (d, 6 H); 13C NMR (75 MHz, DMSO-
obtained as yellowing powder. Mp 198e200 ꢃC; ESI-MS (m/z) 404
[M þ H]þ; ½a 2D0
ꢄ
¼ 55.20 (c 1.3, CH3OH); IR (cmꢁ1): 3403, 3158, 3011,
2929, 1687, 1629, 1515, 1364, 1245, 755; 1H NMR (300 MHz, DMSO-
d6)
d
/ppm ¼ 168.4, 161.7, 142.6, 142.1, 136.0, 130.7, 129.2, 123.0,
d6)
d
/ppm ¼ 13.10 (s, 1 H), 11.99 (s, 1 H), 8.39 (m, 5 H), 7.78 (d,
121.7, 121.2, 114.8, 113.4, 50.1, 23.9, 22.9, 18.6. Anal. Calcd for
C19H23N5O2: C, 64.57; H, 6.56; N, 19.82. Found: C, 64.76; H, 6.73; N,
20.03.
J ¼ 8.4 Hz,1 H), 7.71 (t, J ¼ 7.5 Hz,1 H), 7.41 (t, J ¼ 7.5 Hz,1 H), 7.25 (d,
J ¼ 8.4 Hz, 1 H), 6.73 (m, 4 H), 4.17 (m, 1 H), 4.06 (m, 1 H), 3.20 (m,
1 H), 3.05 (m, 3 H); 13C NMR (75 MHz, DMSO-d6)
d/ppm ¼ 171.5,
168.8,161.1,155.2,142.1,140.2, 133.8,131.7,130.9, 129.0, 128.8, 125.0,
122.4, 121.7, 119.4, 115.8, 61.1, 113.7, 112.3, 53.6, 35.9, 15.9. Anal.
Calcd for C22H21N5O3: C, 65.50; H, 5.25; N, 17.36. Found: C, 65.28; H,
5.10; N, 17.15.
4.2.25. N-(1-methyl-
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3-carbonyl)-N0-glycylhydrazine (6a) from 100 mg
(0.228 mmol) of N-(1-methyl-
-caboline-3-carbonyl)-N0-(Boc-
b
-caboline-3-carbonyl)-N0-valylhydrazine (6e)
b
-
b
valyl)-hydrazine (5e) 71 mg (92%) of the title compound were ob-
4.2.29. N-(1-methyl-b
-caboline-3-carbonyl)-N0-threonylhydrazine
(6i)
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3-carbonyl)-N0-glycylhydrazine (6a) from 100 mg
(0.227 mmol) of N-(1-methyl-
-caboline-3-carbonyl)-N0-(Boc-
tained as yellowing powder. Mp 233e235 ꢃC; ESI-MS (m/z) 340
[M þ H]þ; ½a 2D0
ꢄ
¼ 51.73 (c 1.3, CH3OH); IR (cmꢁ1): 3448, 3068, 2965,
b-
2880, 1715, 1679, 1629, 1507, 1364, 1282, 1249, 1217, 1111, 1035, 869,
755; 1H NMR (300 MHz, DMSO-d6)
d
/ppm ¼ 12.69 (s, 1 H), 10.86 (s,
b
1 H), 9.00 (s, 1 H), 8.40 (m, 4 H), 7.73 (d, J ¼ 7.8 Hz, 1 H), 7.67 (t,
threonyl)-hydrazine (5i) 70 mg (90%) of the title compound were
ꢃ
J ¼ 7.2 Hz, 1 H), 7.37 (t, J ¼ 7.2 Hz, 1 H), 3.78 (m, 1 H), 2.98 (s, 3 H),
obtained as yellowing powder. Mp 214e215 C; ESI-MS (m/z) 342
2.20 (m, 1 H), 1.02 (d, 6 H); 13C NMR (75 MHz, DMSO-d6)
d/
[M þ H]þ; ½a 2D0
ꢄ
¼ 33.33 (c 1.3, CH3OH); IR (cmꢁ1): 3391, 3145, 2978,
ppm ¼ 167.2, 162.0, 142.5, 142.1, 136.1, 130.5, 129.1, 123.0, 121.7,
121.2, 114.7, 113.3, 56.5, 30.4, 18.5. Anal. Calcd for C18H21N5O2: C,
63.70; H, 6.24; N, 22.64. Found: C, 63.91; H, 6.39; N, 22.86.
2924, 1715, 1687, 1629, 1511, 1364, 1278, 1249, 1221, 1111, 1041, 755;
1H NMR (300 MHz, DMSO-d6)
d
/ppm ¼ 12.81 (s, 1 H), 10.95 (s, 2 H),
9.05 (s, 1 H), 8.39 (m, 4 H), 7.76 (d, J ¼ 8.4 Hz, 1 H), 7.69 (t, J ¼ 7.2 Hz,
1 H), 7.39 (t, J ¼ 7.2 Hz, 1 H), 3.97 (m, 1 H), 3.74 (m, 1 H), 3.00 (m,
4.2.26. N-(1-methyl-
(6f)
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3-carbonyl)-N0-glycylhydrazine (6a) from 100 mg
(0.212 mmol) of N-(1-methyl-
-caboline-3-carbonyl)-N0-(Boc-
b
-caboline-3-carbonyl)-N0-methionylhydrazine
3 H), 1.37 (d, J ¼ 6.3 Hz, 1 H); 13C NMR (75 MHz, DMSO-d6)
d/
ppm ¼ 166.3, 162.2, 142.6, 142.1, 136.2, 130.3, 128.9, 122.9, 121.5,
114.5, 113.2, 66.5, 57.7, 20.1. Anal. Calcd for C17H19N5O3: C, 58.81; H,
5.61; N, 20.52. Found: C, 58.60; H, 5.45; N, 20.30.
b-
b
methionyl)-hydrazine (5f) 71 mg (90%) of the title compound were
4.2.30. N-(1-methyl-
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3-carbonyl)-N0-glycylhydrazine (6a) from 100 mg
(0.234 mmol) of N-(1-methyl-
-caboline-3-carbonyl)-N0-(Boc-
b
-caboline-3-carbonyl)-N0-serylhydrazine (6j)
obtained as yellowing powder. Mp 242e243 ꢃC; ESI-MS (m/z) 372
b
-
[M þ H]þ; ½a 2D0
ꢄ
¼ 25.17 (c 1.1, CH3OH); IR (cmꢁ1): 3427, 3080, 2978,
2916, 2843, 1711, 1679, 1629, 1511, 1364, 1249, 1221, 869, 755; 1H
b
NMR (300 MHz, DMSO-d6)
d
/ppm ¼ 12.75 (s,1 H),11.06 (s,1 H), 9.09
seryl)-hydrazine (5j) 73 mg (90%) of the title compound were ob-
(s, 1 H), 8.59 (m, 3 H), 8.37 (d, J ¼ 8.1 Hz,1 H), 7.75 (d, J ¼ 8.1 Hz,1 H),
7.67 (t, J ¼ 8.1 Hz, 1 H), 7.39 (t, J ¼ 7.5 Hz, 1 H), 4.14 (m, 1 H), 3.01 (s,
3 H), 2.71 (m, 2 H), 2.21 (m, 2 H), 2.12 (s, 3 H); 13C NMR (75 MHz,
tained as yellowing powder. Mp 206e208 ꢃC; ESI-MS (m/z) 328
[M þ H]þ; ½a 2D0
ꢄ
¼ 19.23 (c 1.2, CH3OH); IR (cmꢁ1): 3399, 3325, 3146,
3007, 2929, 2851, 1715, 1679, 1629, 1576, 1507, 1364, 1025, 755; 1H
DMSO-d6)
d/ppm ¼ 167.8, 162.5, 142.4, 142.1, 136.2, 130.2, 128.8,
NMR (300 MHz, DMSO-d6)
d/ppm ¼ 12.96 (s, 1 H), 10.98 (s, 1 H),

C. Li et al. / European Journal of Medicinal Chemistry 46 (2011) 5598e5608
5607
10.93 (s, 1 H), 9.06 (s, 1 H), 8.46 (m, 3 H), 8.37 (d, J ¼ 7.8 Hz,1 H), 7.76
(0.183 mmol) of N-(1-methyl-b b-
-caboline-3-carbonyl)-N0-[Boc-(
(d, J ¼ 7.8 Hz, 1 H), 7.69 (t, J ¼ 6.9 Hz, 1 H), 7.39 (t, J ¼ 7.2 Hz, 1 H),
OBzl-aspartyl)]-hydrazine (5n) 40 mg (61%) of the title compound
4.04 (m, 1 H), 3.93 (m, 1 H), 3.83 (m, 1 H), 3.00 (s, 3 H); 13C NMR
were obtained as yellowing powder. Mp 260e261 ꢃC; ESI-MS (m/z)
(75 MHz, DMSO-d6)
d
/ppm ¼ 166.5, 162.3, 142.4, 142.1, 136.2, 130.2,
356 [M þ H]þ; ½a 2D0
ꢄ
¼ 24.97 (c 1.1, CH3OH); IR (cmꢁ1) 3403, 3084,
128.8, 122.9, 121.4, 114.5, 113.2, 61.1, 54.0, 19.2. Anal. Calcd for
C16H17N5O3: C, 58.71; H, 5.23; N, 21.39. Found: C, 58.90; H, 5.38; N,
21.60.
2970, 2921, 2855, 1715, 1683, 1625, 1507, 1364, 1245, 1209, 1143,
759; 1H NMR (300 MHz, DMSO-d6)
d/ppm ¼ 13.12 (s, 1 H), 11.14 (s,
1 H), 10.97 (s, 1 H), 9.12 (s, 1 H), 8.55 (s, 3 H), 8.36 (d, J ¼ 8.1 Hz, 1 H),
7.78 (d, J ¼ 8.1 Hz,1 H), 7.71 (t, J ¼ 7.5 Hz,1 H), 7.41 (t, J ¼ 7.5 Hz,1 H),
4.2.31. N-(1-methyl-
b
-caboline-3-carbonyl)-N0-trptophanylhydrazine
4.28 (m, 1H), 3.02 (m, 5 H); 13C NMR (75 MHz, DMSO-d6)
d/
(6k)
ppm ¼ 171.1, 167.5, 161.8, 142.7, 142.2, 136.0, 133.5, 130.5, 129.1,
123.0, 121.6, 121.2, 114.7, 113.3, 65.3, 48.4, 18.7. Anal. Calcd for
C17H17N5O4: C, 57.46; H, 4.82; N, 19.71. Found: C, 57.24; H, 4.66; N,
19.94.
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3-carbonyl)-N0-glycylhydrazine (6a) from 100 mg
(0.190 mmol) of N-(1-methyl-
-caboline-3-carbonyl)-N0-(Boc-
b-
b
trptophanyl)-hydrazine (5k) 74 mg (91%) of the title compound
were obtained as yellowing powder. Mp 219e220 ꢃC; ESI-MS (m/z)
4.2.35. N-(1-methyl-b g-OBzl-glutamoyl)-
-caboline-3-carbonyl)-N0-(
hydrazine (6o)
427 [M þ H]þ; ½a 2D0
ꢄ
¼ 38.47 (c 1.1, CH3OH); IR (cmꢁ1): 3387, 3145,
2974, 2921, 2847, 1715, 1679, 1625, 1507, 1454, 1364, 1245, 1102, 747;
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3-carbonyl)-N0-glycylhydrazine (6a) from 100 mg
(0.179 mmol) of N-(1-methyl- g-
-caboline-3-carbonyl)-N0-[Boc-(
b-
1H NMR (300 MHz, DMSO-d6)
d
/ppm ¼ 12.65 (s, 1 H), 11.12 (s, 1 H),
8.98 (s, 1 H), 8.36 (m, 3 H), 7.84 (d, J ¼ 7.8 Hz, 1 H), 7.74 (d, J ¼ 8.4 Hz,
1 H), 7.67 (t, J ¼ 7.5 Hz,1 H), 7.40 (m, 3 H), 7.12 (t, J ¼ 7.5 Hz,1 H), 7.05
(t, J ¼ 7.5 Hz,1 H), 4.19 (m, 1 H), 3.43 (dd, J ¼ 5.4 Hz, J ¼ 15.0 Hz, 1 H),
3.25 (dd, J ¼ 8.1 Hz, J ¼ 15.0 Hz, 3 H), 2.99 (s, 3 H); 13C NMR (75 MHz,
b
OBzl-glutamoyl)]-hydrazine (5o) 44 mg (65%) of the title
compound were obtained as yellowing powder. Mp 202e204 ꢃC;
a 20
þ
ESI-MS (m/z) 370 [M þ H] ; ½ ꢄD ¼ 19.67 (c 1.1, CH3OH); IR (cmꢁ1):
DMSO-d6)
d
/ppm ¼ 167.7, 162.1, 142.4, 142.1, 136.8, 136.2, 130.3,
3481, 3101, 2966, 2917, 2851, 1707, 1674, 1629, 1507, 1360, 1249,
128.8, 127.7, 125.8, 123.0, 121.5, 1214, 119.1, 118.9, 114.5, 113.2, 111.9,
107.2, 52.1, 28.0, 19.2. Anal. Calcd for C24H22N6O2: C, 67.59; H, 5.20;
N, 19.71. Found: C, 67.37; H, 5.04; N, 19.48.
1209, 1041, 755; 1H NMR (300 MHz, DMSO-d6)
d/ppm ¼ 13.06 (s,
1 H), 11.18 (s, 1 H), 10.97 (s, 1 H), 9.16 (s, 1 H), 8.60 (s, 3 H), 8.36 (d,
J ¼ 8.1 Hz,1 H), 7.78 (d, J ¼ 8.1 Hz,1 H), 7.71 (t, J ¼ 7.5 Hz,1 H), 7.41 (t,
J ¼ 7.5 Hz, 1 H), 4.04 (m, 1 H), 3.02 (s, 3 H), 2.68 (m, 1 H), 2.11 (m,
4.2.32. N-(1-methyl-
b
-caboline-3-carbonyl)-N0-histidylhydrazine
3 H); 13C NMR (75 MHz, DMSO-d6)
d
/ppm ¼ 173.9, 167.9, 162.1,
(6l)
142.7, 142.2, 136.1, 130.5, 129.1, 123.0, 121.6, 121.3, 114.8, 113.3, 50.9,
29.8, 27.0, 18.7. Anal. Calcd for C18H19N5O4: C, 58.53; H, 5.18; N,
18.96. Found: C, 58.33; H, 5.02; N, 18.74.
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3-carbonyl)-N0-glycylhydrazine (6a) from 100 mg
(0.173 mmol) of N-(1-methyl-
-caboline-3-carbonyl)-N0-(di-Boc-
b-
b
histidyl)-hydrazine (5l) 55 mg (88%) of the title compound were
4.2.36. N-(1-methyl-b
-caboline-3-carbonyl)-N0-(Boc-glutaminyl)-
hydrazine (6p)
obtained as yellowing powder. Mp 195e197 ꢃC; ESI-MS (m/z) 378
[M þ H]þ; ½a 2D0
ꢄ
¼ 32.77 (c 1.1, CH3OH); IR (cmꢁ1): 3415, 3121, 2994,
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3-carbonyl)-N0-glycylhydrazine (6a) from 100 mg
(0.214 mmol) of N-(1-methyl-
-caboline-3-carbonyl)-N0-(Boc-glu-
b-
2921, 2851, 1719, 1683, 1629, 1507, 1364, 1249, 1082, 759; 1H NMR
(300 MHz, DMSO-d6)
d
/ppm ¼ 12.85(s,1 H),11.14 (s,1 H),11.06(s,1 H),
b
9.14 (s, 1 H), 9.04 (s, 1 H), 8.77 (s, 3 H), 8.38 (d, J ¼ 8.4 Hz,1 H), 7.74 (d,
J ¼ 7.8 Hz,1 H), 7.68 (m, 2 H), 7.38 (t, J ¼ 7.5 Hz,1 H), 4.64 (m,1 H), 3.48
taminyl)-hydrazine (5p) 64 mg (81%) of theꢃtitle compound was
obtained as yellowing powder. Mp 212e214 C; ESI-MS (m/z) 369
(m, 2 H), 3.04 (m, 3 H); 13C NMR (75 MHz, DMSO-d6)
d/ppm ¼ 167.1,
[M þ H]þ; ½a 2D0
ꢄ
¼ 46.70 (c 1.1, CH3OH); IR (cmꢁ1): 3415, 3158, 2974,
162.6, 142.5, 142.2, 136.2, 134.5, 130.3, 128.8, 126.6, 122.9, 121.5, 121.3,
118.9,114.6,113.2, 50.7, 26.8, 21.2. Anal. Calcd forC19H19N7O2: C, 60.47;
H, 5.07; N, 25.98. Found: C, 60.26; H, 4.92; N, 25.77.
2921, 2855, 1666, 1625, 1507, 1364, 1245, 1041, 755; 1H NMR
(300 MHz, DMSO-d6)
d
/ppm ¼ 12.93 (s, 1 H), 10.94 (s, 1 H), 9.11 (s,
1 H), 8.58 (s, 3 H), 8.38 (d, J ¼ 8.1 Hz, 1 H), 7.76 (d, J ¼ 8.4 Hz, 1 H),
7.70 (t, J ¼ 8.1 Hz, 1 H), 7.53 (s, 1 H), 7.40 (t, J ¼ 7.5 Hz, 1 H), 7.01 (s,
1 H), 4.01 (m,1 H), 3.02 (s, 3 H), 2.42 (m, 2 H), 2.11 (m, 2 H); 13C NMR
4.2.33. N-(1-methyl-
Using a procedure similar to that of preparing N-(1-methyl-
caboline-3-carbonyl)-N0-glycylhydrazine (6a) from 100 mg
(0.176 mmol) of N-(1-methyl-
-caboline-3-carbonyl)-N0-(di-Boc-
b
-caboline-3-carbonyl)-N0-lysylhydrazine (6m)
b
-
(75 MHz, DMSO-d6)
d
/ppm ¼ 173.9, 167.9, 162.3, 142.4, 142.1, 136.2,
134.4, 130.2, 128.8, 123.0, 121.4, 114.5, 113.2, 51.2, 30.7, 27.7, 19.1.
Anal. Calcd for C18H20N6O3: C, 58.69; H, 5.47; N, 22.81. Found: C,
58.90; H, 5.62; N, 22.58.
b
lysyl)-hydrazine (5m) 59 mg (90%) of the title compound were
obtained as yellowing powder. Mp 192e194 ꢃC; ESI-MS (m/z) 369
[M þ H]þ; ½a 2D0
ꢄ
¼ 42.17 (c 1.2, CH3OH); IR (cmꢁ1): 3472, 2966, 2925,
4.3. In vitro anti-platelet aggregation activity assay
1715, 1679, 1629, 1503, 1364, 1249, 1041, 759; 1H NMR (300 MHz,
DMSO-d6)
d
/ppm ¼ 12.71 (s, 1 H), 10.90 (s, 1 H), 8.53 (s, 3 H), 8.37 (d,
An H-10 cell counter was used to determine the platelet count
and a two-channel Chronolog aggregometer was used to evaluate
platelet aggregation. The pig blood (6 pigs, purchased from Animal
Center of Peking University) was centrifuged at 1000 rpm for
10 min and the platelet rich plasma (PRP) was collected. The
remaining blood was centrifuged for an additional 10 min at
1500 rpm to prepare platelet poor plasma (PPP). The final platelet
count of the PRP was adjusted to 2 ꢂ 108 platelets/ml with autol-
ogous PPP. To an optical aggregometry testing tuber, 0.5 ml of the
J ¼ 7.8 Hz,1 H), 8.15 (s, 3 H), 7.74 (d, J ¼ 8.1 Hz,1 H), 7.68 (t, J ¼ 7.5 Hz,
1 H), 7.38 (t, J ¼ 7.5 Hz,1 H), 3.97 (m, 1 H), 2.99 (s, 3 H), 2.78 (m, 2 H),
1.91 (m, 2 H), 1.60 (m, 4 H); 13C NMR (75 MHz, DMSO-d6)
d/
ppm ¼ 169.4, 161.1, 142.0, 140.3, 133.8, 131.7, 129.0, 128.5, 122.3,
121.7, 119.2, 113.7, 112.1, 52.1, 39.1, 30.4, 26.4, 21.4, 15.8. Anal. Calcd
for C19H24N6O2: C, 61.94; H, 6.57; N, 22.81. Found: C, 61.75; H, 6.41;
N, 22.68.
4.2.34. N-(1-methyl-
hydrazine (6n)
b
-caboline-3-carbonyl)-N0-(
b-OBzl-aspartyl)-
adjusted plasma sample and 5
6aep (in a series of final concentrations of 100, 10, 1, 0.1, 0.01 and
0.001 M, prepared by diluting 10 mM of stock solutions of 6aep in
DMSO/NS, 1/10, with NS) was added. After adjustment of the
mL of NS or 5 mL of the solution of
Using a procedure similar to that of preparing N-(1-methyl-
b
-
m
caboline-3-carbonyl)-N0-glycylhydrazine (6a) from 100 mg

5608
C. Li et al. / European Journal of Medicinal Chemistry 46 (2011) 5598e5608
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baseline, 5 mL of the solution of PAF (final concentration 0.1 mM,
prepared by diluting 10 mM of stock solution of PAF in DMSO/NS, 1/
10, with NS) or 5 L of the solution of ADP (final concentration
10 M, prepared by diluting 10 mM of stock solution of ADP in
DMSO/NS, 1/10, with NS) or 5 L of the solution of arachidonic acid
in NS (AA, final concentration 350 M, prepared by diluting 10 mM
of stock solution of AA in DMSO/NS, 1/10, with NS), or 50 L of the
m
m
m
m
m
solution of TH (final concentration 0.1 U/ml, prepared by diluting
100 U/ml of stock solution of TH in DMSO/NS, 1/10, with NS) was
added and aggregation was measured at 37 ꢃC for 5 min. The effects
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of 6aep (at a series of concentrations ranging from 100 mM to 1 nM)
on PAF or ADP or AA or TH-induced platelet aggregation were
observed. All these anti-platelet aggregation tests in sixplicate
tubers were carried out. The maximum platelet aggregation (Am) of
control group (NS) or sample group (6aep) was represented by the
peak height of aggregation curve (equals to the maximum light
transmission). The inhibition rate was calculated according to the
following formula: Inhibition (%) ¼ [(Am of NS) ꢁ (Am of 6aep)]/(Am
of NS) ꢂ 100%. Am% of NS is the value of platelet aggregation
induced by PAF, ADP, AA and TH without 6aep and are
52.30 ꢀ 1.78%, 50.16 ꢀ 3.65%, 49.62 ꢀ 2.90% and 61.20 ꢀ 2.97%,
respectively. The concentration vs. inhibition rate curve is plotted
to determine the IC50 values with GWBASIC.EXE program.
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4.4. In vivo anti-thrombotic assay
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Acknowledgements
This work was completed in the Beijing area major laboratory of
peptide and small molecular drugs, supported by PHR (IHLB, KZ
200910025004), the National Natural Scientific Foundation of
China (81072522), and Special Project (2011ZX09302-007-01) of
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Appendix. Supplementary material
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.ejmech.2011.09.027.
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